Method for formulating large diameter synthetic membrane vesicles

ABSTRACT

The present invention generally relates to the field of pharmaceutical sciences. More specifically, the present invention includes apparatus and devices for the preparation of pharmaceutical formulations containing large diameter synthetic membrane vesicles, such as multivesicular liposomes, methods for preparing such formulations, and the use of specific formulations for therapeutic treatment of subjects in need thereof. Formation and use of the pharmaceutical formulations containing large diameter synthetic membrane vesicles produced by using the apparatus and devices for therapeutic treatment of subjects in need thereof is also contemplated.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

Any and all priority claims identified in the Application Data Sheet, orany correction thereto, are hereby incorporated by reference under 37CFR 1.57.

FIELD OF THE INVENTION

The present invention generally relates to the field of pharmaceuticalsciences. More specifically, the present invention relates topharmaceutical formulations containing large diameter synthetic membranevesicles, such as multivesicular liposomes (MVL), methods for preparingsuch formulations, and the use of specific formulations for therapeutictreatment of subjects in need thereof.

BACKGROUND

The following includes information that may be useful in understandingthe present embodiments. It is not an admission that any of theinformation provided herein is prior art, or relevant, to the presentlydescribed or claimed embodiments, or that any publication or documentthat is specifically or implicitly referenced is prior art.

Large scale methods of manufacturing large diameter synthetic membranevesicles, such as multivesicular liposomes, often require large amountsof solvents, time sensitive steps and concentration adjustment of thefinal product under sterile conditions. In addition, current methods ofmanufacturing large diameter synthetic membrane vesicles, such asmultivesicular liposomes on a commercial scale, require significantcommitments in manufacturing space, cost, and time. As such, developingstable multivesicular liposome formulations containing a therapeuticagent in a cost effective and timely manner remains an ongoingchallenge.

SUMMARY

Some embodiments provide an atomizing nozzle apparatus, comprising afirst fluid conduit and a second fluid conduit each having at least oneentrance orifice and at least one exit orifice, a fluid contactingchamber having a top comprising at least one entrance orifice and havinga bottom comprising at least one exit orifice and connecting to the atleast one exit orifice of the first fluid conduit, a third liquidchannel, wherein the third fluid conduit annularly surrounds a portionof the fluid contacting chamber. In some embodiments, the fluidcontacting chamber connects to the at least one exit orifice of thesecond fluid conduit. In some embodiments, the at least one exit orificeof the fluid contacting chamber and the at least one exit orifice of thethird fluid conduit are flush. In some embodiments, the at least oneexit orifice of the fluid contacting chamber is recessed within the atleast one exit orifice of the third fluid conduit. In some embodiments,the at least one exit orifice of the fluid contacting chamber extendsbeyond the at least one exit orifice of the third fluid conduit. In someembodiments, the first fluid conduit and the second fluid conduit areco-axial for a first portion of the first fluid conduit length. In someembodiments, the second fluid conduit annularly surrounds a secondportion of the first fluid conduit. In some embodiments, a diameter ofthe fluid contacting chamber is larger than a diameter of the firstfluid conduit. In some embodiments, the fluid contacting chamberconically narrows in diameter from the top of the fluid contactingchamber to the exit orfice of the fluid contacting chamber. In someembodiments, the fluid contacting chamber conically narrows in diameterfrom a point below the top of the fluid contacting chamber to the exitorfice of the fluid contacting chamber.

Some embodiments provide a process for preparing droplets using anatomizing nozzle as disclosed herein comprising applying a first liquidto the first fluid conduit, applying a second liquid to the second fluidconduit, applying a gas to the third fluid conduit, wherein the gasexiting the third fluid conduit exit orifice impinges the liquid exitingthe at least one exit orifice of the fluid contacting chamber, providingatomized droplets, wherein the droplets have an average diameter fromabout 100 nm to about 300 μM. In some embodiments, the first liquid isan emulsion comprised of a first aqueous phase, and a first organicphase comprising a first organic solvent. In some embodiments, the firstorganic solvent is chloroform or methylene chloride. In someembodiments, the first organic phase further comprises at least oneamphipathic lipid and at least one neutral lipid. In some embodiments,the at least one amphipathic lipid is selected from the group consistingof phosphatidylcholines, phosphatidylserines, phosphatidylethanolamines,phosphatidylinositols, sphingomyelin, soybean lecithin (soya lecithin),egg lecithin, lysophosphatidylcholines, lysophosphatidylethanolamines,phosphatidylglycerols, phosphatidylserines, phosphatidylinositols,phosphatidic acids, cardiolipins, acyl trimethylammonium propane,diacyldimethylammonium propane, stearylamine, and ethylphosphatidylcholine. In some embodiments, the at least one amphipathiclipid is selected from the group consisting of1,2-dioleoyl-sn-glycero-3-phosphocholine,1,2-dilauroyl-sn-glycero-3-phosphocholine,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-diarachidoyl-sn-glycero-3-phosphocholine,1,2-dibehenoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine,1,2-dieicosenoyl-sn-glycero-3-phosphocholine,1,2-dierucoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol,1,2-dioleoyl-sn-glycero-3-phosphoglycerol. In some embodiments, the atleast one neutral lipid is selected from the group consisting ofglycerol esters, glycol esters, tocopherol esters, sterol esters,hydrocarbons and squalenes. In some embodiments, the at least oneneutral lipid is selected from the group consisting of triolein,tripalmitolein, trimyristolein, trilinolein, tributyrin, tricaprylin,tricaproin, and tricaprin. In some embodiments, the first aqueous phasefurther comprises a therapeutic agent. In some embodiments, thetherapeutic agent is bupivacaine. In some embodiments, the second liquidapplied to the second fluid conduit is a second aqueous phase. In someembodiments, the droplet comprises a first component core and a secondaqueous phase shell. In some embodiments, the gas is nitrogen. In someembodiments, the droplets have an average diameter from about 20 μM toabout 60 μM. In some embodiments, the droplets have an average diameterfrom about 35 μM to about 45 μM.

Some embodiments provide atomized droplet comprising an emulsion core,wherein the emulsion core comprises i) a first aqueous phase; and ii) afirst organic phase comprising a first organic solvent; and an aqueousphase shell, wherein said atomized droplet is made by a processcomprising combining a first component, an aqueous phase, and a gasusing an atomizing nozzle as disclosed and described herein, saidprocess comprising applying a first component to the first fluidconduit, applying an aqueous phase to the second fluid conduit, andapplying a gas to the third fluid conduit, wherein the gas exiting thethird fluid conduit exit orifice impinges the liquid exiting the atleast one exit orifice of the fluid contacting chamber, providingatomized droplets, wherein the droplets have an average diameter fromabout 100 nm to about 300 μM.

Some embodiments provide atomizing nozzle apparatus, comprising an firstfluid conduit, a second fluid conduit and a third fluid conduit eachhaving at least one entrance orifice and at least one exit orifice, afirst fluid contacting chamber having a top comprising at least oneentrance orifice and having a bottom comprising at least one exitorifice and connecting to the at least one exit orifice of the firstfluid conduit, wherein the second fluid conduit annularly surrounds aportion of the first fluid contacting chamber, a second fluid contactingchamber having a top comprising at least one entrance orifice and havinga bottom comprising at least one exit orifice and connecting to the atleast one exit orifice of the first fluid contacting chamber, whereinthe third fluid conduit annularly surrounds a portion of the secondfluid contacting chamber, and a fourth fluid conduit, wherein the fourthfluid conduit annularly surrounds a portion of the second fluidcontacting chamber. In some embodiments, the first fluid contactingchamber connects to the at least one exit orifice of the second fluidconduit. In some embodiments, the at least one exit orifice of thesecond fluid contacting chamber and the at least one exit orifice of thefourth fluid conduit are flush. In some embodiments, the at least oneexit orifice of the second fluid contacting chamber is recessed withinthe at least one exit orifice of the fourth fluid conduit. In someembodiments, the at least one exit orifice of the second fluidcontacting chamber extends beyond the at least one exit orifice of thefourth fluid conduit. In some embodiments, the first fluid conduit andthe second fluid conduit are co-axial for a first portion of the firstfluid conduits length. In some embodiments, the second fluid conduitannularly surrounds a second portion of the first fluid conduit. In someembodiments, the first fluid contacting chamber conically narrows indiameter from the top of the fluid contacting chamber to the exit orficeof the fluid contacting chamber. In some embodiments, the second fluidconduit and the third fluid conduit are co-axial for a first portion ofthe second fluid conduits length. In some embodiments, the third fluidconduit annularly surrounds a second portion of the second fluidconduit. In some embodiments, the first fluid contacting chamberconically narrows in diameter from the top of the fluid contactingchamber to the exit orfice of the fluid contacting chamber.

Some embodiments provide a process for preparing droplets using anatomizing nozzle apparatus as disclosed and described herein, comprisingapplying a first liquid to the first fluid conduit, applying a secondliquid to the second fluid conduit, applying a third liquid to the thirdfluid conduit, applying a gas to the fourth fluid conduit, wherein thegas exiting the fourth fluid conduit exit orifice impinges the liquidexiting the at least one exit orifice of the second fluid contactingchamber, providing atomized droplets, wherein the droplets have anaverage diameter from about 100 nm to about 300 μM. In some embodiments,the first liquid is an emulsion comprised of i) a first aqueous phase,and ii) a first organic phase comprising a first organic solvent, thesecond liquid is a second aqueous phase, and the third liquid is asecond organic phase. In some embodiments, the first organic solvent ischloroform or methylene chloride. In some embodiments, the first organicphase further comprises at least one amphipathic lipid and at least oneneutral lipid.

Some embodiments provide an evaporation apparatus, comprising at leastone atomizing nozzle apparatus of Claim 1 and means for evaporating anorganic solvent.

Some embodiments provide an evaporation apparatus, comprising a solventremoval vessel having a top, a bottom and a circular wall, at least oneatomizing nozzle connected to the circular wall, a carrier gas entranceorifice connected to the circular wall, a solvent removal gas exitorifice centrally connected to the top, and a product exit orificeconnected to the bottom of the vessel. In some embodiments, at leastpart of the solvent removal vessel is jacketed. In some embodiments, theatomizing nozzle is mounted to and extending through the top of thesolvent removal vessel. In some embodiments, the top of the solventremoval vessel comprises a lid. In some embodiments, the apparatusfurther comprises a rinse nozzle mounted to and extending through thetop of the solvent removal vessel. In some embodiments, the circularwall has a central axis and the solvent removal gas exit orifice furthercomprises a tube extending into the solvent removal vessel residingalong the central axis. In some embodiments, the atomizing nozzle isangled at least 5 degrees measured off the central axis of the wall andin a plane parallel to the wall nearest to it. In some embodiments, thecarrier gas entrance orifice is combined with the atomizing nozzle. Insome embodiments, the solvent removal gas exit orifice further comprisesa tube extending into the solvent removal vessel, wherein the tube isfitted with a narrowing cone and an annular ring. In some embodiments,the tube extends from about ⅓ to about ⅘ of the way into the solventremoval vessel. In some embodiments, the tube extends about ⅔ of the wayinto the solvent removal vessel. In some embodiments, the bottom tip ofthe narrowing cone of the solvent removal gas exit orifice diameter isfrom about 1/1000 to about ⅕ of a diameter of the inside of the solventremoval vessel. In some embodiments, the solvent removal gas exitorifice diameter is less than 1/10 of a diameter of the inside of thesolvent removal vessel. In some embodiments, the at least one atomizingnozzle is an atomizing nozzle apparatus as disclosed and describedherein. In some embodiments, the ratio of the inside diameter of thesolvent removal vessel to the diameter of the bottom tip of thenarrowing cone of the solvent removal gas exit orifice is betweenapproximately 5:1 and 100:1. In some embodiments, the ratio of theinside diameter of the solvent removal vessel to the diameter of thebottom tip of the narrowing cone of the solvent removal gas exit orificeis between approximately 20:1 and 60:1.

Some embodiments provide a process for preparing large diametersynthetic membrane vesicles using an evaporation apparatus as disclosedand described herein, comprising introducing large diameter syntheticmembrane vesicles pre-droplets to the solvent removal vessel, whereinthe large diameter synthetic membrane vesicles pre-droplets comprise afirst component core and an aqueous phase shell, applying a carrier gasin a tangential direction to the circular wall through the carrier gasentrance orifice, and removing a solvent removal gas through the solventremoval gas exit orifice to provide the large diameter syntheticmembrane vesicles. In some embodiments, the first component corecomprises a first aqueous phase and a first organic phase. In someembodiments, the first organic phase comprises a continuous firstorganic solvent. In some embodiments, the first organic solvent ischloroform or methylene chloride. In some embodiments, the first organicphase further comprises at least one amphipathic lipid and at least oneneutral lipid. In some embodiments, the first component core is firstaqueous phase droplets as a suspension in a first organic phase. In someembodiments, the first aqueous phase droplets have an average diameterof from about 10 nm to about 10 μm, about 100 nm to about 5 μm, or about500 nm to about 2 μm. In some embodiments, the first aqueous phasedroplets have an average diameter of about 1 μm. In some embodiments,the carrier gas comprises nitrogen. In some embodiments, the carrier gascomprises nitrogen and water vapor. In some embodiments, the solventremoval gas comprises nitrogen and organic solvent. In some embodiments,the carrier gas and the solvent removal gas travel in a vortex in thesolvent removal vessel. In some embodiments, the large diametersynthetic membrane vesicles are multivesicular liposomes having astructure including multiple non-concentric chambers and comprising atleast one amphipathic lipid and at least one neutral lipid. In someembodiments, the at least one amphipathic lipid is selected from thegroup consisting of phosphatidylcholines, phosphatidylserines,phosphatidylethanolamines, phosphatidylinositols, sphingomyelin, soybeanlecithin (soya lecithin), egg lecithin, lysophosphatidylcholines,lysophosphatidylethanolamines, phosphatidylglycerols,phosphatidylserines, phosphatidylinositols, phosphatidic acids,cardiolipins, acyl trimethylammonium propane, diacyldimethylammoniumpropane, stearylamine, and ethyl phosphatidylcholine. In someembodiments, the at least one amphipathic lipid is selected from thegroup consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine,1,2-dilauroyl-sn-glycero-3-phosphocholine,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-diarachidoyl-sn-glycero-3-phosphocholine,1,2-dibehenoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine,1,2-dieicosenoyl-sn-glycero-3-phosphocholine,1,2-dierucoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, and1,2-dioleoyl-sn-glycero-3-phosphoglycerol. In some embodiments, the atleast one neutral lipid is selected from the group consisting ofglycerol esters, glycol esters, tocopherol esters, sterol esters,hydrocarbons and squalenes. In some embodiments, the at least oneneutral lipid is selected from the group consisting of triolein,tripalmitolein, trimyristolein, trilinolein, tributyrin, tricaprylin,tricaproin, and tricaprin. In some embodiments, the multivesicularliposomes further comprises a therapeutic agent. In some embodiments,the therapeutic agent is bupivacaine. In some embodiments, themultivesicular liposomes comprises an outer surface layer whosecomposition is different than the composition of the internal structure.

Some embodiments provide an evaporation apparatus, comprising at leastone atomizing nozzle apparatus as disclosed and described herein andmeans for removing an organic solvent from a droplet.

Some embodiments provide a process for preparing large diametersynthetic membrane vesicles using the evaporation apparatus as disclosedand described herein, comprising introducing large diameter syntheticmembrane vesicles pre-droplets to the solvent removal vessel, whereinthe large diameter synthetic membrane vesicles pre-droplets comprise afirst component core and an aqueous phase shell; applying a carrier gasin a tangental direction to the circular wall through the carrier gasentrance orifice; removing a solvent removal gas through the solventremoval gas exit orifice to provide pre-temperature treatment largediameter synthetic membrane vesicles; introducing the pre-temperaturetreatment large diameter synthetic membrane vesicles to an outlet line;contacting the pre-temperature treatment large diameter syntheticmembrane vesicles with a hot solution in the outlet line, wherein thehot solution has a temperature ranging from about 30° C. to about 100°C. to provide post-temperature treatment large diameter syntheticmembrane vesicles; transferring the post-temperature treatment largediameter synthetic membrane vesicles to a continuous-flowparticle-concentration system or continuous phase exchange system;cooling the post-temperature treatment large diameter synthetic membranevesicles to a second temperature to provide the large diameter syntheticmembrane vesicles; and isolating the large diameter synthetic membranevesicles. In some embodiments, the first component core comprises afirst aqueous phase and a first organic phase. In some embodiments, thefirst organic phase comprises a continuous first organic solvent. Insome embodiments, the first organic solvent is chloroform or methylenechloride. In some embodiments, the first organic phase further comprisesat least one amphipathic lipid and at least one neutral lipid. In someembodiments, the first component core is a suspension of first aqueousphase droplets in a first organic phase. In some embodiments, the firstaqueous phase droplets have an average diameter of from about 10 nm toabout 10 μm, about 100 nm to about 5 μm, or about 500 nm to about 2 μm.In some embodiments, the first aqueous phase droplets have an averagediameter of about 1 μm. In some embodiments, the carrier gas comprisesnitrogen. In some embodiments, the solvent removal gas comprisesnitrogen and organic solvent. In some embodiments, the carrier gas andthe solvent removal gas travel in a vortex in the solvent removalvessel. In some embodiments, the large diameter synthetic membranevesicles are multivesicular liposomes having a structure includingmultiple non-concentric chambers and comprising at least one amphipathiclipid and at least one neutral lipid. In some embodiments, the at leastone amphipathic lipid is selected from the group consisting ofphosphatidylcholines, phosphatidylserines, phosphatidylethanolamines,phosphatidylinositols, sphingomyelin, soybean lecithin (soya lecithin),egg lecithin, lysophosphatidylcholines, lysophosphatidylethanolamines,phosphatidylglycerols, phosphatidylserines, phosphatidylinositols,phosphatidic acids, cardiolipins, acyl trimethylammonium propane,diacyldimethylammonium propane, stearylamine, and ethylphosphatidylcholine. In some embodiments, the at least one amphipathiclipid is selected from the group consisting of1,2-dioleoyl-sn-glycero-3-phosphocholine,1,2-dilauroyl-sn-glycero-3-phosphocholine,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-diarachidoyl-sn-glycero-3-phosphocholine,1,2-dibehenoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine,1,2-dieicosenoyl-sn-glycero-3-phosphocholine,1,2-dierucoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, and1,2-dioleoyl-sn-glycero-3-phosphoglycerol. In some embodiments, the atleast one neutral lipid is selected from the group consisting ofglycerol esters, glycol esters, tocopherol esters, sterol esters,hydrocarbons and squalenes. In some embodiments, the at least oneneutral lipid is selected from the group consisting of triolein,tripalmitolein, trimyristolein, trilinolein, tributyrin, tricaprylin,tricaproin, and tricaprin. In some embodiments, the multivesicularliposomes further comprises a therapeutic agent. In some embodiments,the therapeutic agent is bupivacaine.

Some embodiments provide a composition comprising multivesicularliposomes having a structure including multiple non-concentric chambersand comprising at least one amphipathic lipid and at least one neutrallipid, wherein said multivesicular liposomes are made by a processcomprising removing organic solvent from multivesicular liposomespre-droplets using an evaporation apparatus as disclosed and describedherein, said process comprising introducing multivesicular liposomespre-droplets pre-droplets to the solvent removal vessel, wherein thelarge diameter synthetic membrane vesicles pre-droplets comprise a firstcomponent core and an aqueous phase shell; applying a carrier gas in atangental direction to the circular wall through the carrier gasentrance orifice; and removing a solvent removal gas through the solventremoval gas exit orifice to provide the large diameter syntheticmembrane vesicles.

Some embodiments provide a composition comprising large diametersynthetic membrane vesicles made by a process disclosed and describedherein. In some embodiments, the large diameter synthetic membranevesicles are multivesicular liposomes having a structure includingmultiple non-concentric chambers and comprising at least one amphipathiclipid and at least one neutral lipid. In some embodiments, the at leastone amphipathic lipid is selected from the group consisting ofphosphatidylcholines, phosphatidylserines, phosphatidylethanolamines,phosphatidylinositols, sphingomyelin, soybean lecithin (soya lecithin),egg lecithin, lysophosphatidylcholines, lysophosphatidylethanolamines,phosphatidylglycerols, phosphatidylserines, phosphatidylinositols,phosphatidic acids, cardiolipins, acyl trimethylammonium propane,diacyldimethylammonium propane, stearylamine, and ethylphosphatidylcholine. In some embodiments, the at least one amphipathiclipid is selected from the group consisting of1,2-dioleoyl-sn-glycero-3-phosphocholine,1,2-dilauroyl-sn-glycero-3-phosphocholine,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-diarachidoyl-sn-glycero-3-phosphocholine,1,2-dibehenoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine,1,2-dieicosenoyl-sn-glycero-3-phosphocholine,1,2-dierucoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, and1,2-dioleoyl-sn-glycero-3-phosphoglycerol. In some embodiments, the atleast one neutral lipid is selected from the group consisting ofglycerol esters, glycol esters, tocopherol esters, sterol esters,hydrocarbons and squalenes. In some embodiments, the at least oneneutral lipid is selected from the group consisting of triolein,tripalmitolein, trimyristolein, trilinolein, tributyrin, tricaprylin,tricaproin, and tricaprin. In some embodiments, the multivesicularliposomes further comprises a therapeutic agent. In some embodiments,the therapeutic agent is bupivacaine.

Some embodiments provide a continuous-flow emulsification system,comprising a mixer, comprised of a rotor and a stator; a recirculationloop, comprised of one or more recirculation lines; a heat exchanger;one or more outlet lines; one or more continuous phase inlet lines; anda discontinuous phase inlet line, wherein the heat exchanger and themixer are connected together in the recirculation loop by one or morerecirculation lines; further wherein the one or more outlet lines andone or more continuous phase inlet lines are connected to therecirculation loop; further wherein the end of the discontinuous phaseinlet line is located within approximately ⅓rd of a rotor diameter fromthe rotor and approximately ⅓rd of a rotor diameter of the rotation axisof the rotor and is in fluid communication with the rotor. In someembodiments, a continuous phase entrance line is connected to therecirculation loop upstream of the mixer and downstream of the heatexchanger and an outlet line is connected to the recirculation loopdownstream of the mixer and upstream of the heat exchanger. In someembodiments, the continuous-flow emulsification system further comprisesan emulsion, which emulsion recirculates through the recirculation lineback to the mixer an average of at least 5 or more times. In someembodiments, the continuous-flow emulsification system further comprisesemulsion droplets produced in the mixer, which are on average less than10 microns in diameter.

Some embodiments provide a continuous processing system, comprising oneor more concentrator units, each unit comprising a retentate vessel; aparticle suspension inlet line, connected to the retentate vessel; afirst outlet line, connecting the retentate vessel and a particleconcentrating device; a pump located along the first outlet line betweenthe retentate vessel and the particle concentrating device; and a secondoutlet line, leading to another concentrator unit or the final productcollection vessel; and means for removing or exchanging solvent. In someembodiments, the means for removing or exchanging solvent are eachindependently selected from the group consisting of a tangential flowfiltration unit, a hydro-cyclone unit, and a centrifugal separator. Insome embodiments, the continuous processing system further comprises anew suspending medium inlet line connected to the retentate vessel. Insome embodiments, the means for removing or exchanging solvent are eacha tangential flow filtration unit. In some embodiments, the means forremoving or exchanging solvent are each a centrifugal separator. In someembodiments, the system comprises at least one tangential flowfiltration unit and at least one centrifugal separator. In someembodiments, the continuous processing system is a continuous-flowparticle-concentration system or continuous phase exchange system.

Some embodiments provide a process for making multivesicular liposomesusing the atomizing nozzle apparatus as disclosed and described herein,comprising applying a first liquid to the first fluid conduit, whereinthe first liquid comprises an organic solvent, applying a second liquidto the second fluid conduit, applying a pressurized gas to the thirdfluid conduit to provide atomized droplets, wherein the pressurized gasexiting the third fluid conduit exit orifice impinges the liquid exitingthe fluid contacting chamber exit orifice, and removing the organicsolvent from the atomized droplets, wherein less than 4000 ppm of theorganic solvent remains in the atomized droplets. In some embodiments,the first liquid is an emulsion comprised of a discontinuous aqueousphase, and a continuous organic phase comprising the organic solvent. Insome embodiments, the organic solvent is methylene chloride. In someembodiments, the continuous organic phase further comprises atherapeutic agent. In some embodiments, the therapeutic agent isbupivacaine or a salt thereof. In some embodiments, the second liquidapplied to the second fluid conduit is an aqueous solution. In someembodiments, the aqueous solution further comprises dextrose and lysine.In some embodiments, the gas is a sterilized gas. In some embodiments,the gas is nitrogen. In some embodiments, the process further comprisesintroducing atomized droplets to an evaporation apparatus as disclosedand described herein; introducing a pressurized carrier gas tangentiallyto the circular wall into the solvent removal vessel through the carriergas entrance orifice; removing a solvent removal gas wherein the solventremoval gas removes greater than 90% of the organic solvent in theatomized droplets resulting in formation of multivesicular liposomes. Insome embodiments, the carrier gas is heated and humidified. In someembodiments, the process further comprises spraying a wall rinsesolution into the solvent removal vessel using a rinse nozzle, whereinthe wall rinse solution prevents build-up of particles in theevaporation apparatus. In some embodiments, the atomized dropletscontain organic solvent in the range of from about 400 ppm to about 3500ppm.

Some embodiments provide a process for making an emulsion using anemulsification system as disclosed and described herein, comprisingfeeding an organic discontinuous phase into the emulsification systemthrough the discontinuous phase inlet line and feeding an aqueouscontinuous phase into the emulsification system through one or morecontinuous phase inlet lines.

Some embodiments provide a process for making an emulsion using theemulsification system as disclosed and described herein, comprisingfeeding an aqueous discontinuous phase into the emulsification systemthrough the discontinuous phase inlet line and feeding an organiccontinuous phase fed into the emulsification system through the one ormore continuous phase inlet lines. In some embodiments, the organiccontinuous phase is comprised of an organic solvent and a neutral lipid.In some embodiments, the organic solvent is methylene chloride. In someembodiments, the aqueous discontinuous phase is comprised of an acid anda therapeutic agent. In some embodiments, the acid is phosphoric acid.In some embodiments, the therapeutic agent is bupivacaine. In someembodiments, a portion of the emulsion is fed through one or more outletlines to the inner fluid conduit of an atomizing nozzle as disclosed anddescribed herein. In some embodiments, a portion of the emulsion is fedthrough one or more outlet lines to an evaporation apparatus asdisclosed and described herein.

Some embodiments provide a process for preparing large diametersynthetic membrane vesicles using an evaporation apparatus as disclosedand described herein.

Some embodiments provide a large diameter synthetic membrane vesiclesmade by a process as disclosed and described herein.

Some embodiments produce a plurality of MVL particles made by using theprocess utilizing the atomizing nozzle disclosed herein.

Some embodiments produce a plurality of MVL particles made by using theprocess utilizing the emulsification system and the atomizing nozzledisclosed herein.

Some embodiments produce a plurality of MVL particles made by using theprocess utilizing the evaporation apparatus and the atomizing nozzledisclosed herein.

Some embodiments produce a plurality of MVL particles made by using theprocess utilizing the emulsification system, the evaporation apparatusand the atomizing nozzle disclosed herein.

Some embodiments produce a plurality of MVL particles made by using theprocess utilizing the evaporation apparatus, the atomizing nozzle, andparticle concentration system disclosed herein.

Some embodiments produce a plurality of MVL particles made by using theprocess utilizing the emulsification system, the evaporation apparatus,the atomizing nozzle, and particle concentration system disclosedherein.

Some embodiments produce a plurality of MVL particles made by any of theabove product-by-process embodiments, wherein the MVL containsbupivacaine or a pharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects and features of the instant embodiment willbecome more fully apparent in the following descriptions and appendedclaims taken in conjuction with the following drawings where likereferences numbers indicate identical or functionally similar elements.

FIG. 1A is a schematic diagram of significant components used in one ofthe instant systems for manufacturing synthetic membrane vesicles.

FIG. 1B is a schematic diagram of significant components used in anothersystem for manufacturing synthetic membrane vesicles.

FIG. 1C is a schematic of one embodiment of a continuous heat treatmentsystem including a temperature controlled tank and a holding coil tubingused in a manufacturing system such as the manufacturing system of FIG.1A or FIG. 1B.

FIG. 2 is a schematic of one embodiment of an emulsification system usedin the manufacturing system of FIG. 1A or FIG. 1B.

FIG. 3A is a schematic view of one embodiment of a three channelatomizing nozzle used in a manufacturing system, such as themanufacturing system of FIG. 1A of FIG. 1B.

FIG. 3B is a cross-sectional view of the atomizing nozzle of FIG. 3A,taken along line B of FIG. 3A.

FIG. 3C is an expanded view of the cylindrical tip of the atomizingnozzle of FIG. 3A, shown as position C in FIG. 3A.

FIG. 3D is an expanded view of an atomized droplet produced by theatomizing nozzle of FIG. 3A, shown as position D in FIG. 3A.

FIG. 3E is a schematic view of one embodiment of a four channelatomizing nozzle used in a manufacturing system, such as themanufacturing system of FIG. 1A or FIG. 1B.

FIG. 3F is a cross-sectional view of the atomizing nozzle of FIG. 3E,taken along line F of FIG. 3E.

FIG. 3G is an expanded view of the cylindrical tip of the atomizingnozzle of FIG. 3E, shown as position G in FIG. 3E.

FIG. 3H is an expanded view of an atomized droplet produced by theatomizing nozzle of FIG. 3E, shown as position H in FIG. 3E.

FIG. 3I is a schematic view of another embodiment of a four channelatomizing nozzle used in a manufacturing system, such as themanufacturing system of FIG. 1A or FIG. 1B.

FIG. 3J is a cross-sectional view of the atomizing nozzle of FIG. 3I,taken along line J of FIG. 3I.

FIG. 3K is an expanded view of the cylindrical tip of the atomizingnozzle of FIG. 3I, shown as position K in FIG. 3I.

FIG. 3L is an expanded view of an atomized droplet produced by theatomizing nozzle of FIG. 3I, shown as position L in FIG. 3I.

FIG. 4A is a schematic view of one embodiment of an atomizing nozzleused in a manufacturing system, such as the manufacturing system of FIG.1A or FIG. 1B.

FIG. 4B is a cross-sectional view of the atomizing nozzle of FIG. 4A,taken along line B of FIG. 4A.

FIG. 4C is an expanded view of the cylindrical tip of the atomizingnozzle of FIG. 4A, shown as position C in FIG. 4A.

FIG. 4D is an expanded view of an atomized droplet produced by theatomizing nozzle of FIG. 4A, shown as position D in FIG. 4A.

FIG. 5 is a detailed view of one embodiment of an atomizing nozzle usedin a manufacturing system, such as the manufacturing system of FIG. 1Aor FIG. 1B.

FIG. 6 is an exploded view of the individual components of the atomizingnozzle of FIG. 5.

FIG. 7 is a schematic of one embodiment of a solvent removal vessel usedfor evaporating solvent from atomized particles in a manufacturingsystem such as the manufacturing system of FIG. 1A or FIG. 1B.

FIG. 8 is a schematic of one embodiment of a particle concentrationsystem used in a manufacturing system such as the manufacturing systemof FIG. 1A or FIG. 1B.

FIG. 9A and FIG. 9B provide cross-sectional views of a droplet, a moredetailed view of a droplet, and a large diameter synthetic membranevesicle particle. In some embodiments, the large diameter syntheticmembrane vesicle particle can be a MVL particle. The large diametersynthetic membrane vesicle particle can be formed by removal of theorganic solvent from the emulsion droplet.

FIG. 10 is a schematic of one embodiment of a particle concentrationsystem including a plurality of filtration units and at least onecentrifuge unit used in a manufacturing system such as the manufacturingsystem of FIG. 1A or FIG. 1B.

FIG. 11 is a schematic of one embodiment of a particle concentrationsystem including a plurality of centrifuge units used in a manufacturingsystem such as the manufacturing system of FIG. 1A or FIG. 1B.

FIG. 12 is a graph of an in vitro release study of multivesicularliposome formulations.

FIG. 13 is a graph of an in vivo PK study of multivesicular liposomeformulations.

FIG. 14 is a graph of an accelerated stability profile study of amultivesicular formulation made with the instant manufacturing systemwith and without heat treatment. The low osmolality is more stable thanthe sample without heat treatment and the heat treated sample is moststable.

FIG. 15 is a graph of an in-vivo release profile study of amultivesicular formulation made with and without heat treatment.

Large scale methods of manufacturing large diameter synthetic membranevesicles, such as multivesicular liposomes, often require large amountsof solvents, time sensitive steps and concentration adjustment of thefinal product under sterile conditions, for example, as described inWO99/25319. In addition, current methods of manufacturing large diametersynthetic membrane vesicles, such as multivesicular liposomes on acommercial scale, require significant commitments in manufacturingspace, cost, and time. As such, developing stable multivesicularliposome formulations containing a therapeutic agent in a cost effectiveand timely manner remains an ongoing challenge.

In large scale manufacturing of large diameter synthetic membranevesicles, the present embodiments require less water for injection(WFI), space, time and energy to produce an equivalent amount of thelarge diameter synthetic membrane vesicles than under previouslydescribed large scale manufacturing conditions. As a result, wastedisposal is reduced and overall costs are reduced. For example, thepresent embodiments provide systems that can be housed in a one storyroom (e.g. 5×5 meter room) that previously described systems wouldrequire a multistory room with at least a ten fold increase in the areaof the room. Additionally, the present embodiments provide systems thatare particularly well adapted to continuous processing for more rapidproduction of large diameter synthetic membrane vesicles and allowingfor more efficient implementation of clean-in-place (CIP) andsterile-in-place (SIP) protocols. Allowing for greatly reduced utilityrequirements to implement CIP and SIP protocols.

The devices described in FIGS. 1A and 1B are particularly well suitedfor making multivesicular liposomes (MVL). Multivesicular liposomes(MVL), first reported by Kim, et al. (Biochim, Biophys. Acta,728:339-348, 1983), are uniquely different from other lipid-based drugdelivery systems such as unilamellar (Huang, Biochemistry, 8:334-352,1969; Kim, et al., Biochim. Biophys. Acta, 646:1-10, 1981) andmultilamellar (Bangham, et al., J. Mol. Bio., 13:238-252, 1965)liposomes. For example, multivesicular liposomes made by the processesdescribed herein typically can have diameters ranging from about 10 to100 μm, and more typically ranging from about 20 to 55 μm. In contrast,multilamellar liposomes usually have diameters of from 0.2 to 5 μm andunilamellar liposomes usually have diameters of from 0.02 to 0.5 μm.

Additionally, multivesicular liposomes (MVL) contain multiple aqueouschambers per particle and the multiple aqueous chambers arenon-concentric. In contrast unilamellar liposomes (also known asunilamellar vesicles) and multilamellar liposomes (also known asmultilamellar vesicles) contain a single chamber per particle. Further,neutral lipids are necessary to form multivesicular liposomes (MVL). Incontrast unilamellar liposomes (also known as unilamellar vesicles) andmultilamellar liposomes (also known as multilamellar vesicles) do notrequire inclusion of neutral lipids to form.

Multivesicular liposomes (MVL) are entirely distinct from unilamellarliposomes and multilamellar liposomes. The structural and functionalcharacteristics of multivesicular liposomes are not directly predictablefrom current knowledge of unilamellar liposomes and multilamellarliposomes. As described in the book edited by Jean R. Philippot andFrancis Schuber (Liposomes as Tools in Basic Research and Industry, CRCpress, Boca Raton, Fla., 1995, page 19), Multivesicular liposomes (MVL)are bounded by an external bilayer membrane shell, but have a verydistinctive internal morphology, which may arise as a result of thespecial method employed in the manufacture. Topologically,multivesicular liposomes (MVL) are defined as liposomes containingmultiple non-concentric chambers within each liposome particle,resembling a “foam-like” matrix; whereas multilamellar vesicles containmultiple concentric chambers within each liposome particle, resemblingthe “layers of an onion”.

The presence of internal membranes distributed as a network throughoutmultivesicular liposomes (MVL) may serve to confer increased mechanicalstrength to the vesicle, while still maintaining a high volume:lipidratio compared with multilamellar vesicles. The multivesicular nature ofmultivesicular liposomes (MVL) also indicates that, unlike forunilamellar liposomes, a single breach in the external membrane of amultivesicular liposomes (MVL) will not result in total release of theinternal aqueous contents. Thus, both structurally and functionally themultivesicular liposomes (MVL) are unusual, novel and distinct from allother types of liposomes. As a result, the functional properties ofmultivesicular liposomes (MVL) are not predictable based on the priorart related to conventional liposomes such as unilamellar liposomes andmultilamellar liposomes.

In some embodiments, the large diameter synthetic membrane vesicles aremultivesicular liposomes. In some embodiments, the multivesicularliposomes further comprise bupivaciane, DEPC, DPPG, and tricaprylin. Insome embodiments, the multivesicular liposomes further comprisebupivacaine phosphate, DEPC, DPPG, and tricaprylin. In some embodiments,the multivesicular liposomes further comprise bupivacaine, DEPC, DPPG,tricaprylin and cholesterol. In some embodiments, the multivesicularliposomes further comprise bupivacaine phosphate, DEPC, DPPG,tricaprylin and cholesterol.

In some embodiments, the multivesicular liposomes further comprisebupivaciane, dextrose, L-Lysine, DEPC, DPPG, and tricaprylin. In someembodiments, the multivesicular liposomes further comprise bupivacainephosphate, dextrose, L-Lysine, DEPC, DPPG, and tricaprylin. In someembodiments, the multivesicular liposomes further comprise bupivacaine,dextrose, L-Lysine, DEPC, DPPG, tricaprylin and cholesterol. In someembodiments, the multivesicular liposomes further comprise bupivacainephosphate, dextrose, L-Lysine, DEPC, DPPG, tricaprylin and cholesterol.

In some embodiments, the multivesicular liposomes further comprisebupivaciane, dextrose, DEPC, DPPG, and tricaprylin. In some embodiments,the multivesicular liposomes further comprise bupivacaine phosphate,dextrose, DEPC, DPPG, and tricaprylin. In some embodiments, themultivesicular liposomes further comprise bupivacaine, dextrose, DEPC,DPPG, tricaprylin and cholesterol. In some embodiments, themultivesicular liposomes further comprise bupivacaine phosphate,dextrose, DEPC, DPPG, tricaprylin and cholesterol.

In some embodiments, the multivesicular liposomes further comprisebupivaciane, L-Lysine, DEPC, DPPG, and tricaprylin. In some embodiments,the multivesicular liposomes further comprise bupivacaine phosphate,L-Lysine, DEPC, DPPG, and tricaprylin. In some embodiments, themultivesicular liposomes further comprise bupivacaine, L-Lysine, DEPC,DPPG, tricaprylin and cholesterol. In some embodiments, themultivesicular liposomes further comprise bupivacaine phosphate,L-Lysine, DEPC, DPPG, tricaprylin and cholesterol.

In some embodiments, the multivesicular liposomes further comprisebupivacaine, morphine, cytarabine, or their pharmaceutically acceptablesalts as the therapeutic agent. In some embodiments, the multivesicularliposomes further comprise bupivacaine phosphate, morphine sulfate, orcytarabine HCl.

In another embodiment, any one of the above described embodiments can beused alone or in combination with any one or more of the above describedembodiments. For example, any above described atomizing nozzle,evaporation apparatus, continuous-flow emulsification system,continuous-flow diafiltration system, continuous-flow diafiltrationfurther comprising one or more centrifuges, continuous-flow centrifugesystem, or continuous processing system can be used alone or incombination. Thus, an evaporation apparatus can be used in conjunctionwith a three-fluid atomizing nozzle. This evaporation system/atomizingnozzle can be used with a continuous-flow emulsification system, asdepicted in FIGS. 1A, 1B, and 1C. The three-fluid atomizingnozzle/evaporation apparatus combination can be used in conjunction witha continuous-flow system, as depicted in FIGS. 8, 10, and 11. Any ofthese combinations can be used to make multivesicular liposomes. Inparticular any of the combinations can be used to make multivesicularliposomes containing bupivacaine or its salts as the therapeutic agent.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Features of the present disclosure will become more fully apparent fromthe following description and appended claims, taken in conjunction withthe accompanying drawings. It will be understood these drawings depictonly certain embodiments in accordance with the disclosure and,therefore, are not to be considered limiting of its scope; thedisclosure will be described with additional specificity and detailthrough use of the accompanying drawings. An apparatus, system or methodaccording to some of the described embodiments can have several aspects,no single one of which necessarily is solely responsible for thedesirable attributes of the apparatus, system or method. Afterconsidering this discussion, and particularly after reading the sectionentitled “Detailed Description of the Preferred Embodiment” one willunderstand how illustrated features serve to explain certain principlesof the present disclosure.

In the following detailed description, only certain exemplaryembodiments have been shown and described, simply by way ofillustration. As those skilled in the art would realize, the describedembodiments may be modified in various different ways, all withoutdeparting from the spirit or scope of the present disclosure.Accordingly, the drawings and description are to be regarded asillustrative in nature and not restrictive. In addition, when an elementis referred to as being “on” another element, it can be directly on theanother element or be indirectly on the another element with one or moreintervening elements interposed therebetween. Also, when an element isreferred to as being “connected to” another element, it can be directlyconnected to the another element or be indirectly connected to theanother element with one or more intervening elements interposedtherebetween. Hereinafter, like reference numerals refer to likeelements. Since the disclosure may be modified in various ways and havevarious embodiments, the disclosure will be described in detail withreference to the drawings. However, it should be understood that thedisclosure is not limited to a specific embodiment but includes allchanges and equivalent arrangements and substitutions included in thespirit and scope of the disclosure. In the following description, if thedetailed description of the already known structure and operation mayconfuse the subject matter of the present disclosure, the detaileddescription thereof will be omitted.

While such terms as “first,” “second,” etc., may be used to describevarious components, such components must not be limited to the aboveterms. The above terms are used only to distinguish one component fromanother. Terms used in the following description are to describespecific embodiments and is not intended to limit the disclosure. Theexpression of singularity includes plurality meaning unless thesingularity expression is explicitly different in context. It should beunderstood that the terms “comprising,” “having,” “including,” and“containing” are to indicate features, numbers, steps, operations,elements, parts, and/or combinations but not to exclude one or morefeatures, numbers, steps, operations, elements, parts, and/orcombinations or additional possibilities.

Some embodiments provide continuous processes for making multivesicularliposomes. Prior methods of making multivesicular liposomes requiredbatch processing. This batch processing required removal of the solventfrom the droplets of first emulsion surrounded by a second aqueous phaseby contacting the suspension of first emulsion droplets in a continuousaqueous phase with a discontinuous gas phase by sparging (bubbling) gasthrough the aqueous phase or blowing gas over a flask containingcontinuous aqueous phase. This batch processing takes tens of minutes toremove the solvent.

It was surprisingly discovered that forming a first emulsion surroundedby an aqueous shell in the form of a droplet and contacting it with acontinuous gas phase, reduces the time needed to remove the organicsolvent to a few seconds and possibly a fraction of a second. (much lessthan the tens of minutes stated above for batch processing).

This is due to the tremendous gas contacting surface area of theatomized droplets; and the much faster diffusion of the solvent ingasses versus water; and the very short distances that the solvent hasto diffuse through the aqueous phase to reach the gas (now only micronsinstead of the distanced between sparging bubbles.)

This extremely fast solvent removal enables the continuous processing.The solvent is removed in less than the time that it takes for theatomized droplets to reach the bottom of the solvent removal vessel (afew seconds at most).

DEFINITIONS

As used herein, abbreviations are defined as follows:

-   aq. Aqueous-   CIP Clean-in-place processing-   ° C. Temperature in degrees Celsius-   d10 the diameter where 10 mass-% (volume %) (of the particles) of    the particles have a smaller equivalent diameter, and the other 90    mass-% (volume %) have a larger equivalent diameter in μm-   d50 the diameter where 50 mass-% (volume %) (of the particles) of    the particles have a smaller equivalent diameter, and the other 50    mass-% (volume %) have a larger equivalent diameter in μm-   d90 the diameter where 90 mass-% (volume %) (of the particles) of    the particles have a smaller equivalent diameter, and the other 100    mass-% (volume %) have a larger equivalent diameter in μm-   DCM methylene chloride-   g Gram(s)-   h Hour (hours)-   mL Milliliter(s)-   mg Milligram(s)-   mOsm/kg Osmolality per kilogram-   pH measure of the acidity or alkalinity of a liquid using a pH meter    or pH indicator-   PPV Packed particle volume-   PSD Particle size distribution-   RT, rt Room temperature-   SIP Sterile-in-place processing-   Tert, t tertiary-   μL Microliter(s)-   μg Microgram(s)-   WFI Water for injection

Some embodiments relate to a process for preparing a large diametersynthetic membrane vesicle composition comprising preparing a firstcomponent by mixing a first aqueous phase and an organic phase, saidorganic phase comprising an organic solvent, at least one amphipathiclipid and at least one neutral lipid; preparing a droplet(s) by mixingsaid first component and a second aqueous phase, said droplet(s)comprising an aqueous phase; preparing a large diameter syntheticmembrane vesicle by removing the organic solvent from the droplet(s),wherein the removing comprises contacting the droplet(s) with a gas; andcollecting the large diameter synthetic membrane vesicle particles,wherein the large diameter synthetic membrane vesicle is suspended in acontinuous aqueous phase.

Some embodiments relate to a process for preparing a multivesicularliposome composition comprising preparing a first component by mixing afirst aqueous phase and an organic phase, said organic phase comprisingan organic solvent, at least one amphipathic lipid and at least oneneutral lipid, wherein the first component comprises a therapeuticagent; preparing a droplet(s) by mixing said first component and asecond aqueous phase, said emulsion droplet(s) comprising an aqueousphase; preparing a multivesicular liposome particle by removing theorganic solvent from the emulsion droplet, wherein the removingcomprises contacting the emulsion droplet with a gas; and preparing amultivesicular liposome composition by collecting the multivesicularliposome particles, wherein the multivesicular liposome composition issuspended in a continuous aqueous phase.

Some embodiments relate to a process for preparing a multivesicularliposome composition comprising preparing a first component by mixing afirst aqueous phase and an organic phase, said organic phase comprisingan organic solvent, at least one amphipathic lipid and at least oneneutral lipid, wherein the first component comprises a therapeuticagent, preparing an emulsion of first component droplets, a secondcomponent droplet, by mixing said first component and a second aqueousphase, said second component droplet comprising an aqueous phase,wherein the second component droplet is prepared using a device asdescribed herein, preparing a multivesicular liposome particle byremoving the organic solvent from the second component droplet, whereinthe removing comprises contacting the second component droplet with agas, and preparing a multivesicular liposome composition by collectingthe multivesicular liposome particles, wherein the multivesicularliposome composition is suspended in a continuous aqueous phase.

As used herein the term “amphipathic lipid” refers to a substanceincluding a hydrophilic region and a hydrophobic region, such asphospholipids. Amphipathic lipids can be zwitterionic phospholipids,zwitterionic lipids, lipids having a net negative charge, and lipidshaving a net positive charge. For example, amphipathetic lipids include,but are not limited to, phosphatidylcholines, phosphatidylserines,phosphatidylethanolamines, phosphatidylinositols, sphingomyelin, soybeanlecithin (soya lecithin), egg lecithin, lysophosphatidylcholines,lysophosphatidylethanolamines, phosphatidylglycerols,phosphatidylserines, phosphatidylinositols, phosphatidic acids,cardiolipins, acyl trimethylammonium propane, diacyldimethylammoniumpropane, stearylamine, ethyl phosphatidylcholine and the like.Phospholipids used in the methods described herein can be of a singleclass or a mixture of classes. Some embodiments include crudepreparations of phospholipids, such as soybean lecithin (soya lecithin)and egg lecithin. Soya lecithin is a combination predominantly ofnaturally-occurring phospholipids; phosphatidylcholine (PC),phosphatidylethanolamine (PE) and phosphatidylinositol (PI). Examples ofphosphatidylcholines and phosphatidylglycerols include, but are notlimited to, 1,2-dioleoyl-sn-glycero-3-phosphocholine,1,2-dilauroyl-sn-glycero-3-phosphocholine,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-diarachidoyl-sn-glycero-3-phosphocholine,1,2-dibehenoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine,1,2-dieicosenoyl-sn-glycero-3-phosphocholine,1,2-dierucoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol,1,2-dioleoyl-sn-glycero-3-phosphoglycerol, and mixtures thereof.

As used herein the term “neutral lipid” refers to oils, waxes or fattyacid esters that lack a charged or hydrophilic head group. Neutrallipids include but are not limited to, glycerol esters, glycol esters,tocopherol esters, sterol esters, hydrocarbons and squalenes.

As used herein the terms “glycerol esters,” “triglycerides,” and“triacylglycerols” refer to triesters formed from glycerol and fattyacids. Glycerol esters include but are not limited to, triolein,tripalmitolein, trimyristolein, trilinolein, tributyrin, tricaprylin,tricaproin, and tricaprin.

As used herein the term “organic solvent” refers to ethers, esters,halogenated ethers, aromatic or aliphatic hydrocarbons, aromatic oraliphatic halohydrocarbons, or Freons. Organic solvents include but arenot limited to, diethyl ether, tert-butylmethyl ether, tetrahydrofuran,sevoflurane, desflurane, isoflurane, enflurane, halothane, chloroform,dichloromethane, ethyl acetate, hexane, hexanes, cyclohexane, pentane,cyclopentane, petroleum ether, toluene, and any combinations thereof.

As used herein the term “aqueous phase” refers to any solution ormixture having water as the major component. The aqueous phase caninclude constituents, such as pH buffering agents, salts, osmoticagents, simple sugars, amino acids, electrolytes, preservatives, otherwater soluble excipients and the like. Aqueous phase constituents caninclude, but are not limited to, sodium chloride, hydrochloric acid,phosphoric acid, lysine, dextrose, glucose and the like.

As used herein the term “fluid” refers to a substance that has theability to flow. Fluids can be a gas, a liquid, a liquid with asubstance(s) suspended throughout the liquid, an emulsion, a vapor, or agas/vapor mixture.

As used herein the term “therapeutic agent” and “drug” refers to achemical compound, mixtures of chemical compounds, or biologicalmolecules, such as biological macromolecules or peptides that may havetherapeutic properties. The therapeutic agent can be purified,substantially purified or partially purified. In some embodiments, thetherapeutic agent can be selected from the group including antianginas,antiarrhythmics, antiasthmatic agents, antibiotics, antidiabetics,antifungals, antihistamines, antihypertensives, antiparasitics,antineoplastics, antitumor drugs, antivirals, cardiac glycosides,hormones, immunomodulators, monoclonal antibodies, neurotransmitters,nucleic acids, proteins, radio contrast agents, radionuclides,sedatives, analgesics, steroids, tranquilizers, vaccines, vasopressors,anesthetics, peptides and the like. Any pharmaceutically acceptable saltof a particular therapeutic agent is also envisioned as being useful inthe present embodiments. In some embodiments, the therapeutic agent canbe introduced in either an aqueous or a solvent phase, depending ontheir solubility in these phases. In a typical embodiment, thetherapeutic agent can be selected from the group including semisyntheticaminoglycoside antibiotics such as amikacin; antidiabetics; peptidessuch as insulin; antitumor drugs such as paclitaxel; antineoplasticsincluding cytarabine, 5-fluorouracil and floxuridine; alkaloid opiateanalgesics including morphine and hydromorphine; local anestheticsincluding bupivacaine; synthetic anti-inflamniatory adrenocorticalsteroids including dexamethasone; antimetabolites includingmethotrexate; glycopeptide antibiotics including bleomycin;vincaleukoblastines and stathmokinetic oncolytic agents includingvincristine and vinblastine; hormones, plasma proteins, cytokines,growth factors, DNA and RNA from a variety of organisms, and antisenseoligonucleotides. In some embodiments, the therapeutic agent can be anamide anesthetic. Amide anesthetics include, but are not limited to,bupivacaine, mepivacaine, ropivacaine, lidocaine, pyrrocaine,prilocaine, their stereoisomers, and combinations thereof, andpharmaceutically acceptable salts thereof.

As used herein the term “pharmaceutically acceptable salt” refers to asalt of a compound that does not cause significant irritation to anorganism to which it is administered and does not abrogate thebiological activity and properties of the compound. In some embodiments,the salt is an acid addition salt of the compound. Pharmaceutical saltscan be obtained by reacting a compound with inorganic acids such ashydrohalic acid (e.g., hydrochloric acid or hydrobromic acid), sulfuricacid, nitric acid, phosphoric acid and the like. Pharmaceutical saltscan also be obtained by reacting a compound with an organic acid such asaliphatic or aromatic carboxylic or sulfonic acids, for example acetic,succinic, lactic, malic, tartaric, citric, ascorbic, nicotinic,methanesulfonic, ethanesulfonic, p-toluensulfonic, salicylic ornaphthalenesulfonic acid. Pharmaceutical salts can also be obtained byreacting a compound with a base to form a salt such as an ammonium salt,an alkali metal salt, such as a sodium or a potassium salt, an alkalineearth metal salt, such as a calcium or a magnesium salt, a salt oforganic bases such as dicyclohexylamine, N-methyl-D-glucamine,tris(hydroxymethyl)methylamine, C₁-C₇ alkylamine, cyclohexylamine,triethanolamine, ethylenediamine, and salts with amino acids such asarginine, lysine, and the like. In some embodiments, the therapeuticagent can have a low aqueous solubility in neutral form. In someembodiments, the pharmaceutically acceptable salt of a therapeutic agentcan have higher aqueous solubility in comparison to a therapeutic agentin neutral form.

Many and varied therapeutic agents can be incorporated by encapsulationwithin the synthetic membrane vesicles. A non-limiting list oftherapeutic agent classes include, but are not limited to, antianginas,antiarrhythmics, antiasthmatic agents, antibiotics, antidiabetics,antifungals, antihistamines, antihypertensives, antiparasitics,antineoplastics, antiobesity agents, antiviral agents, otologicals,cardiac glycosides, hormones, immunomodulators, monoclonal antibodies,neurotransmitters, sedatives, vaccines, vasopressors, anesthetics, amideanaesthetics, corticosteroids, tricyclic antidepressants, tetracyclicantidepressants, selective serotonin reuptake inhibitors, steroidreceptor modulators, antipsychotic drugs, antiprotozoal drugs, opioids,antiproliferative agents, salicylanilides, antihelminthic drugs, vincaalkaloids, anti-inflammatory agents, antidepressants, prostaglandins,phosphodiesterase IV inhibitors; retinoids, steroids, β-adrenergicreceptor ligands, anti-mitotic agents, microtubule inhibitors,microtubule-stabilizing agents, serotonin norepinephrine reuptakeinhibitors, noradrenaline reuptake inhibitors, non-steroidalimmunophilin-dependent immunosuppressants, non-steroidalimmunophilin-dependent immunosuppressant enhancers; antimalarial agents,analgesics, immunosuppressants, expectorants, sulfa drugs,cardiovascular drugs, central nervous system (CNS) depressants,H2-blockers, anti-platelet drugs, anticonvulsants, alpha blockers,beta-blockers, cholinesterase inhibitors, calcium channel blockers,H1-receptor antagonists, and proteinaceous materials. The therapeuticagents listed herein can be used in the preparation of medicaments forthe treatment of a disease for which the therapeutic agent is known tothose of skill in the art to be effective. Therapeutic agents, anddiseases for which the therapeutic agent is effective, can be identifiedby reference to, for example, The Physician's Desk Reference, which isincorporated herein by reference in its entirety.

Examples of proteinaceous materials that can be incorporated into thesynthetic membrane vesicles, include but are not limited to, DNA, RNA,proteins of various types, protein hormones produced by recombinant DNAtechnology effective in humans, hematopoietic growth factors, monokines,lymphokines, tumor necrosis factor, inhibin, tumor growth factor alphaand beta, Mullerian inhibitory substance, nerve growth factor,fibroblast growth factor, platelet-derived growth factor, pituitary andhypophyseal hormones including LH and other releasing hormones.

Examples of antiarrhythmics, include but are not limited to, quinidine,procainamide, disopyramide, ajmaline, lidocaine, tocamide, mexiletine,flecainide, propafenone, moricizine, propranolol, esmolol, timolol,metoprolol, atenolol, amiodarone, sotalol, ibutilide, dofetilide,verapamil, diltiazem, and digoxin.

Examples of antiasthmatic agents, include but are not limited to,salbutamol, levalbuterol, terbutaline, bitolterol, epinephrine,ipratropium bromide, salmeterol, formoterol, bambuterol, and albuterol.

Examples of antibiotics, include but are not limited to, amikacin,gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin,paromomycin, geldanamycin, herbimycin, loracarbef, ertapenem, doripenem,imipenem, meropenem, cefadroxil, cefazolin, cefalotin, cefalexin,cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime,cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime,ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime,ceftobiprole, teicoplanin, vancomycin, azithromycin, clarithromycin,dirithromycin, erythromycin, roxithromycin, troleandomycin,telithromycin, spectinomycin, aztreonam, amoxicillin, ampicillin,azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin,mezlocillin, meticillin, nafcillin, oxacillin, penicillin, piperacillin,ticarcillin, bacitracin, colistin, polymyxin b, ciprofloxacin, enoxacin,gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin,ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, mafenide,prontosil, sulfacetamide, sulfamethizole, sulfanilimide, sulfasalazine,sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole,demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline,arsphenamine, chloramphenicol, clindamycin, lincomycin, ethambutol,fosfomycin, fusidic acid, furazolidone, isoniazid, linezolid,metronidazole, mupirocin, nitrofurantoin, platensimycin, pyrazinamide,quinupristin, rifampin, thiamphenicol, and timidazole.

Examples of antidiabetics, include but are not limited to, tolbutamide,acetohexamide, tolazamide, chlorpropamide, glipizide, glyburide,glimepiride, gliclazide, repaglinide, nateglinide, metformin,rosiglitazone, pioglitazone, troglitazone, miglitol, acarbose,exenatide, liraglutide, taspoglatide, vildagliptin, sitagliptin, GLP-1,and analog to GLP-1.

Examples of antifungals, include but are not limited to, natamycin,rimocidin, filipin, nystatin, amphotericin B, candicin, miconazole,ketoconazole, clotrimazole, econazole, bifonazole, butoconazole,fenticonazole, isoconazole, oxiconazole, sertaconazole, sulconazole,tioconazole, fluconazole, itraconazole, isavuconazole, ravuconazole,posaconazole, voriconazole, terconazole, abafungin, terbinafine,amorolfine, naftifine, butenafine, anidulafungin, caspofungin,micafungin, ciclopirox, tolnaftate, undecylenic acid, 5-fluorocytosine,and griseofulvin.

Examples of antihistamines, include but are not limited to,aceprometazine, alimemazine, astemizole, azatadine, azelastine,benadryl, bepotastine, bisulepine, brompheniramine, chlorcyclizine,chloropyramine, chlorothen, chlorphenamine, cinnarizine, clemastine,clemizole, clobenzepam, clobenztropine, clocinizine, cyclizine,cyproheptadine, dacemazine, dexbrompheniramine, dexchlorpheniramine,diphenhydramine, doxylamine, drixoral, ebastine, embramine, emedastine,epinastine, etymemazine, fexofenadine, homochlorcyclizine, hydroxyzine,iproheptine, isopromethazine, ketotifen, levocabastine, mebhydrolin,mepyramine, methafurylene, methapyrilene, methdilazine, moxastine,p-methyldiphenhydramine, pemirolast, pheniramine, phenyltoloxamine,resporal, rondec, semprex-d, setastine, sominex, talastine, terfenadine,thenyldiamine, thiazinamium, and triprolidine.

Examples of antihypertensives, include but are not limited to,bumetanide, ethacrynic acid, furosemide, torsemidet, epitizide,hydrochlorothiazide, chlorothiazide, bendroflumethiazide, indapamide,chlorthalidone, metolazone, amiloride, triamterene, spironolactone,atenolol, metoprolol, nadolol, oxprenolol, pindolol, propranolol,timolol, doxazosin, phentolamine, indoramin, phenoxybenzamine, prazosin,terazosin, tolazoline, bucindolol, carvedilol, labetalol, clonidine,methyldopa, guanfacine, amlodipine, felodipine, isradipine,lercanidipine, nicardipine, nifedipine, nimodipine, nitrendipine,diltiazem, verapamil, captopril, enalapril, fosinopril, lisinopril,perindopril, quinapril, ramipril, trandolapril, benazepril, candesartan,eprosartan, irbesartan, losartan, olmesartan, telmisartan, valsartan,eplerenone, spironolactone, sodium nitroprus side, clonidine, guanabenz,methyldopa, moxonidine, guanethidine, and reserpine.

Examples of antiparasitics, include but are not limited to, mebendazole,pyrantel pamoate, thiabendazole, diethycarbazine, niclosamide,praziquantel, rifampin, amphotericin B, and melarsoprol.

Examples of antineoplastics, include but are not limited to,aclarubicin, altretamine, aminopterin, amrubicin, azacitidine,azathioprine, belotecan, busulfan, camptothecin, capecitabine,carboplatin, carmofur, carmustine, chlorambucil, cisplatin, cladribine,clofarabine, cyclophosphamide, cytarabine, daunorubicin, decitabine,doxorubicin, epirubicin, etoposide, floxuridine, fludarabine,5-fluorouracil, fluorouracil, gemcitabine, idarubicin, ifosfamide,irinotecan, mechlorethamine, melphalan, mercaptopurine, methotrexate,mitoxantrone, nedaplatin, oxaliplatin, pemetrexed, pentostatin,pirarubicin, pixantrone, procarbazine, pyrimethamine raltitrexed,rubitecan, satraplatin, streptozocin, thioguanine, triplatintetranitrate, teniposide, topotecan, tegafur, trimethoprim, uramustine,vairubicin, vinblastine, vincristine, vindesine, vinflunine,vinorelbine, and zorubicin.

Examples of antiviral agents, include but are not limited to, abacavir,aciclovir, acyclovir, adefovir, amantadine, amprenavir, arbidol,atazanavir, atripla, boceprevir, cidofovir, combivir, darunavir,delavirdine, didanosine, edoxudine, efavirenz, emtricitabine,enfuvirtide, entecavir, famciclovir, fomivirsen, fosamprenavir,foscarnet, fosfonet, ganciclovir, ibacitabine, immunovir, idoxuridine,imiquimod, indinavir, inosine, lamivudine, lopinavir, loviride,maraviroc, moroxydine, nelfinavir, nevirapine, nexavir, oseltamivir,penciclovir, peramivir, pleconaril, raltegravir, ribavirin, rimantadine,ritonavir, saquinavir, stavudine, tenofovir, tenofovir disoproxil,tipranavir, trifluridine, trizivir, tromantadine, valaciclovir,valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine,zanamivir, and zidovudine.

Examples of otologicals, include but are not limited to, betamethasone,chloramphenicol, chlorhexidine, clioquinol, dexamethasone, gentamicin,hydrocortisone, lidocaine, miconazole, neomycin, nitrofural, polymyxinb, prednisolone, rifamycin, and tetracycline.

Examples of cardiac glycosides, include but are not limited to,digitoxin, digoxin, and deslanoside.

Examples of hormones, include but are not limited to, adiponectin,adrenocorticotropic hormone, aldosterone, androstenedione,angiotensinogen, angiotensin, antidiuretic hormone, antimullerianhormone, atrial-natriuretic peptide, brain natriuretic peptide,25-hydroxyvitamin D₃, calcitonin, 1,25-dihydroxyvitamin D₃,cholecystokinin, corticotropin-releasing hormone, cortisol,dehydroepiandrosterone, dihydrotestosterone, dopamine, endothelin,enkephalin, epinephrine, erythropoietin, estradiol, estriol, estrone,follicle-stimulating hormone, gastrin, ghrelin, glucagon,gonadotropin-releasing hormone, growth hormone, growth hormone-releasinghormone, histamine, human chorionic gonadotropin, human placentallactogen, inhibin, insulin, insulin-like growth factor, leptin,leukotrienes, lipotropin, luteinizing hormone, melanocyte stimulatinghormone, melatonin, neuropeptide y, norepinephrine, orexin, oxytocin,pancreatic polypeptide, parathyroid hormone, progesterone, prolactin,prolactin releasing hormone, prostacyclin, prostaglandins, relaxin,renin, secretin, serotonin, somatostatin, testosterone, thrombopoietin,thromboxane, thyroid-stimulating hormone, thyrotropin-releasing hormone,thyroxine, and triiodothyronine.

Examples of immunomodulators, include but are not limited to, abatacept,abetimus, adalimumab, afelimomab, aflibercept, afutuzumab, alefacept,anakinra, aselizumab, atlizumab, atorolimumab, azathioprine,basiliximab, belatacept, belimumab, bertilimumab, cedelizumab,clenoliximab, certolizumab pegol, ciclosporin, daclizumab, deforolimus,dorlimomab aritox, dorlixizumab, efalizumab, erlizumab, elsilimomab,etanercept, everolimus, faralimomab, fontolizumab, galiximab,gantenerumab, gavilimomab golimumab, gomiliximab, gusperimus,infliximab, inolimomab, ipilimumab keliximab, lebrilizumab, leflunomide,lenalidomide, lerdelimumab, lumiliximab, maslimomab, mepolizumab,metelimumab, methotrexate, morolimumab, muromonab-cd3, mycophenolicacid, natalizumab, nerelimomab, ocrelizumab, odulimomab, omalizumab,otelixizumab, pascolizumab, pexelizumab, pimecrolimus, reslizumab,rilonacept, rovelizumab, ruplizumab, siplizumab, sirolimus, tacrolimus,talizumab, telimomab aritox, temsirolimus, teneliximab, teplizumab,teriflunomide, thalidomide, tocilizumab, toralizumab, tremelimumab,ustekinumab, vapaliximab, vepalimomab, visilizumab, zanolimumab,ziralimumab, zolimomab aritox, zotarolimus, and tetrachlorodecaoxide.

Examples of monoclonal antibodies, include but are not limited to,abagovomab, abatacept, abciximab, adalimumab, adecatumumab, aflibercept,afutuzumab, alacizumab pegol, alemtuzumab, altumomab, afelimomab,anatumomab mafenatox, anrukinzumab, apolizumab, arcitumomab, aselizumab,atlizumab, atorolimumab, bapineuzumab, basiliximab, bavituximab,bectumomab, belatacept, belimumab, bertilimumab, besilesomab,bevacizumab, biciromab brallobarbital, bivatuzumab mertansine,blinatumomab, briakinumab, canakinumab, cantuzumab mertansine, capromabpendetide, catumaxomab, cedelizumab, certolizumab pegol, cetuximab,citatuzumab bogatox, cixutumumab, clenoliximab, golimumab, ustekinumab,conatumumab, dacetuzumab, dacliximab, daclizumab, denosumab, detumomab,dorlimomab aritox, dorlixizumab, ecromeximab, eculizumab, edobacomab,edrecolomab, efalizumab, efungumab, elsilimomab, enlimomab pegol,epitumomab cituxetan, epratuzumab, erlizumab, ertumaxomab, etanercept,etaracizumab, exbivirumab, fanolesomab, faralimomab, felvizumab,fezakinumab, figitumumab, fontolizumab, foravirumab, galiximab,gantenerumab, gavilimomab, gemtuzumab ozogamicin, golimumab,gomiliximab, ibalizumab, ibritumomab tiuxetan, igovomab, imciromab,infliximab, intetumumab, inolimomab, inotuzumab ozogamicin, ibalizumab,ipilimumab, iratumumab, keliximab, labetuzumab, lemalesomab,lebrilizumab, lerdelimumab, lexatumumab, libivirumab, lintuzumab,lucatumumab, lumiliximab, mapatumumab, maslimomab, matuzumab,mepolizumab, metelimumab, milatuzumab, minretumomab, mitumomab,morolimumab, motavizumab, muromonab, stamulumab, nacolomab tafenatox,naptumomab estafenatox, natalizumab, nebacumab, necitumumab,nerelimomab, nimotuzumab, nofetumomab merpentan, ocrelizumab,odulimomab, ofatumumab, omalizumab, oportuzumab monatox, oregovomab,otelixizumab, pagibaximab, palivizumab, panitumumab, panobacumab,pascolizumab, pemtumomab, pertuzumab, pexelizumab, pintumomab,priliximab, pritumumab, rafivirumab, ramucirumab, ranibizumab,raxibacumab, regavirumab, reslizumab, rilonacept, rilotumumab,rituximab, robatumumab, rovelizumab, rozrolimupab, ruplizumab,satumomab, sevirumab, sibrotuzumab, siltuximab, siplizumab, solanezumab,sonepcizumab, sontuzumab, stamulumab, sulesomab, tacatuzumab tetraxetan,tadocizumab, talizumab, tanezumab, taplitumomab paptox, tefibazumab,telimomab aritox, tenatumomab, teneliximab, teplizumab, ticilimumab,tigatuzumab, tocilizumab, toralizumab, tositumomab, trastuzumab,tremelimumab, tucotuzumab celmoleukin, tuvirumab, urtoxazumab,ustekinumab, vapaliximab, vedolizumab, veltuzumab, vepalimomab,visilizumab, volociximab, votumumab, zalutumumab, zanolimumab,ziralimumab, and zolimomab aritox.

Examples of neurotransmitters, include but are not limited to,acetylcholine, adenosine, adenosine-5′-triphosphate, aspartate,norepinephrine, dopamine, glycine, serotonin, melatonin, histamine,glutamate, gamma aminobutyric acid, and guanosine-5′-triphosphate.

Examples of sedatives, include but are not limited to, alprazolam,amobarbital, carisoprodol, chlordiazepoxide, clomethiazole, clonazepam,diazepam, diphenhydramine, estazolam, eszopiclone, ethchlorvynol,flunitrazepam, gamma-hydroxybutyrate, glutethimide, ketamine, lorazepam,methaqualone, methyprylon, midazolam, nitrazepam, oxazepam,pentobarbital, phenobarbitoltriazolam, ramelteon, secobarbital,temazepam, thalidomide, zaleplon, zolpidem, and zopiclone.

Examples of vaccines, include but are not limited to, measles vaccine,mumps vaccine, rubella vaccine, varicella vaccine, inactivated poliovaccine, inactivated influenza vaccine, influenza a virus subtype H1N1vaccine, diphtheria toxoid vaccine, tetanus toxoid vaccine, haemophilusinfluenzae type B vaccine, hepatitis B vaccine, hepatitis A vaccine, andpneumoccocal conjugate vaccine.

Examples of vasopressors, include but are not limited to, epinephrine,phenylephrine, dobutamine, isoproterenol, norepinephrine,aceprometazine, alimemazine, astemizole, azatadine, azelastine,benadryl, bepotastine, bisulepine, brompheniramine, chlorcyclizine,chloropyramine, chlorothen, chlorphenamine, cinnarizine, clemastine,clemizole, clobenzepam, clobenztropine, clocinizine, cyclizine,cyproheptadine, dacemazine, dexbrompheniramine, dexchlorpheniramine,diphenhydramine, doxylamine, drixoral, ebastine, embramine, emedastine,epinastine, etymemazine, fexofenadine, homochlorcyclizine, hydroxyzine,iproheptine, isopromethazine, ketotifen, levocabastine, mebhydrolin,mepyramine, methafurylene, methapyrilene, methdilazine, moxastine,p-methyldiphenhydramine, pemirolast, pheniramine, phenyltoloxamine,resporal, rondec, semprex-d, setastine, sominex, talastine, terfenadine,thenyldiamine, thiazinamium, and triprolidine.

Examples of anesthetics, include but are not limited to, propofol,etomidate, methohexital and sodium thiopental, midazolam, diazepam, andketamine, benzocaine, chloroprocaine, cocaine, cyclomethycaine,dimethocaine, propoxycaine, procaine, proparacaine, tetracaine,articaine, bupivacaine, carticaine, dibucaine, etidocaine,levobupivacaine, lidocaine, mepivacaine, piperocaine, prilocaine,ropivacaine, trimecaine, saxitoxin, and tetrodotoxin.

Examples of amide anesthetics, include but are not limited to,articaine, bupivacaine, carticaine, dibucaine, etidocaine,levobupivacaine, lidocaine, mepivacaine, piperocaine, prilocaine,ropivacaine, and trimecaine.

Examples of corticosteroids, include but are not limited to,hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortolpivalate, prednisolone, methylprednisolone, prednisone, triamcinoloneacetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide,desonide, fluocinonide, fluocinolone acetonide, halcinonide,betamethasone, betamethasone sodium phosphate, dexamethasone,dexamethasone sodium phosphate, fluocortolone,hydrocortisone-17-butyrate, hydrocortisone-17-valerate, aclometasonedipropionate, betamethasone valerate, betamethasone dipropionate,prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate,fluocortolone caproate, fluocortolone pivalate, and fluprednideneacetate.

Examples of tricyclic antidepressants, include but are not limited to,amitriptyline, butriptyline, clomipramine, dosulepin, doxepin,imipramine, lofepramine, trimipramine, desipramine, nortriptyline, andprotriptyline.

Examples of tetracyclic antidepressants, include but are not limited to,amoxapine, maprotiline, mianserin, mirtazapine, and setiptiline.

Examples of selective serotonin reuptake inhibitors, include but are notlimited to, citalopram, dapoxetine, escitalopram, fluoxetine,fluvoxamine, paroxetine, sertraline, vilazodone, and zimelidine.

Examples of antipsychotic drugs, include but are not limited to,haloperidol, droperidol, chlorpromazine, fluphenazine, perphenazine,prochlorperazine, thioridazine, trifluoperazine, mesoridazine,periciazine, promazine, triflupromazine, levomepromazine, promethazine,pimozide, chlorprothixene, flupenthixol, thiothixene, zuclopenthixol,clozapine, olanzapine, risperidone, quetiapine, ziprasidone,amisulpride, asenapine, paliperidone, aripiprazole, and bifeprunox.

Examples of antiprotozoal drugs, include but are not limited to,eflornithine, furazolidone, melarsoprol, metronidazole, ornidazole,paromomycin sulfate, pentamidine, pyrimethamine, and timidazole.

Examples of opioids, include but are not limited to, endorphins,enkephalins, dynorphins, endomorphins, codeine, morphine, thebaine,oripavine, diacetylmorphine, dihydrocodeine, hydrocodone, hydromorphone,nicomorphine, oxycodone, oxymorphone, fentanyl, alphamethylfentanyl,alfentanil, sufentanil, remifentanil, carfentanyl, ohmefentanyl,pethidine, ketobemidone, allylprodine, prodine, propoxyphene,dextropropoxyphene, dextromoramide, bezitramide, piritramide, methadone,dipipanone, levomethadyl acetate, loperamide, diphenoxylate, dezocine,pentazocine, phenazocine, buprenorphine, dihydroetorphine, etorphine,butorphanol, nalbuphine, levorphanol, levomethorphan, lefetamine,meptazinol, tilidine, tramadol, tapentadol, nalmefene, naloxone, andnaltrexone.

Examples of antiproliferative agents, include but are not limited to,aclarubicin, altretamine, aminopterin, amrubicin, azacitidine,azathioprine, belotecan, busulfan, camptothecin, capecitabine,carboplatin, carmofur, carmustine, chlorambucil, cisplatin, cladribine,clofarabine, cyclophosphamide, cytarabine, daunorubicin, decitabine,docetaxel, doxorubicin, epirubicin, etoposide, floxuridine, fludarabine,5-fluorouracil, fluorouracil, gemcitabine, idarubicin, ifosfamide,irinotecan, mechlorethamine, melphalan, mercaptopurine, methotrexate,mitoxantrone, nedaplatin, oxaliplatin, paclitaxel, pemetrexed,pentostatin, pirarubicin, pixantrone, procarbazine, pyrimethamineraltitrexed, rubitecan, satraplatin, sirolimus, streptozocin,thioguanine, triplatin tetranitrate, teniposide, topotecan, tegafur,trimethoprim, uramustine, valrubicin, vinblastine, vincristine,vindesine, vinflunine, vinorelbine, and zorubicin.

Examples of salicylanilides, include but are not limited to,niclosamide, oxyclozanide, and rafoxanide.

Examples of antihelminthic drugs, include but are not limited to,abamectin, albendazole, diethylcarbamazine, mebendazole, niclosamide,ivermectin, suramin, thiabendazole, pyrantel pamoate, levamisole,praziquantel, triclabendazole, flubendazole, fenbendazole, emodepside,and monepantel.

Examples of vinca alkaloids, include but are not limited to,vinblastine, vincristine, vindesine and vinorelbine.

Examples of anti-inflammatory agents, include but are not limited to,phenylbutazone, mofebutazone, oxyphenbutazone, clofezone, kebuzone,indometacin, sulindac, tolmetin, zomepirac, diclofenac, alclofenac,bumadizone, etodolac, lonazolac, fentiazac, acemetacin, difenpiramide,oxametacin, proglumetacin, ketorolac, aceclofenac, bufexamac, piroxicam,tenoxicam, droxicam, lornoxicam, meloxicam, ibuprofen, naproxen,ketoprofen, fenoprofen, fenbufen, benoxaprofen, suprofen, pirprofen,flurbiprofen, indoprofen, tiaprofenic acid, oxaprozin, ibuproxam,dexibuprofen, flunoxaprofen, alminoprofen, dexketoprofen, mefenamicacid, tolfenamic acid, flufenamic acid, meclofenamic acid, celecoxib,rofecoxib, valdecoxib, parecoxib, etoricoxib, lumiracoxib, nabumetone,niflumic acid, azapropazone, glucosamine, benzydamine, glucosaminoglycanpolysulfate, proquazone, orgotein, nimesulide, feprazone, diacerein,morniflumate, tenidap, oxaceprol, and chondroitin sulfate.

Examples of cancers that can be treated with an anticancer agentinclude, but are not limited to, head and neck cancer, breast cancer,colorectal cancer, gastric cancer, hepatic cancer, bladder cancer,cervical cancer, endometrial cancer, lung cancer (non-small cell),ovarian cancer, pancreatic cancer, prostate cancer; choriocarcinoma(lung cancer); hairy cell leukemia, chronic lymphotic leukemia, acutelymphocytic leukemia (breast & bladder), acute myelogenous leukemia,Hodgkin's lymphoma, non-Hodgkin's lymphoma (osteogenic sarcoma, adultsoft tissue sarcoma), meningeal leukemia, multiple myeloma, chronicmyelogenous leukemia, erythroleukemia, and T-cell lymphoma.

Examples of inflammatory and autimmune diseases that can be treated withan inflammatory agent include, but are not limited to, B cell disorders,T cell disorders, rheumatoid arthritis (RA), systemic lupuserythematosus (SLE), Sjogren's syndrome, immune thrombocytopenic purpura(ITP), multiple sclerosis (MS), myasthenia Gravis (MG), Graves disease,psoriasis, Hashimoto's disease, immune thrombocytopenic purpura,scleroderma, and inflamatory bowel disease (e.g. Crohn's disease andulcerative colitis).

In some embodiments, the therapeutic agent can be used singly or incombination with the limitation that the amount of the physiologicallyactive substance in the pharmaceutical composition be sufficient toenable the diagnosis of, prophylaxis against, or treatment of anundesired condition in a living being. In some embodiments, thepharmaceutical compositions can be administered to a living being by anydesired route, for example, intramuscular, intra articular, epidural,intraperitoneal, subcutaneous, intra lymphatic, oral, submucosal,transdermal, rectal, vaginal, intranasal, intraocular, and byimplantation under different kinds of epithelia, including the bronchialepithelia, the gastrointestinal epithelia, the urogenital epithelia, andthe various mucous membranes of the body. Generally, the dosage willvary with the age, condition, sex and extent of the undesired conditionin the patient, and can be determined by one skilled in the art. In someembodiments, the dosage range appropriate for human use includes a rangeof from 0.1 to 6,000 mg of the therapeutic agent per square meter ofsurface area. Alternate dosage range can be based on weight instead ofsurface area. In one embodiment, a human dosage of bupivacaine can be50-1,000 mg, 100-600 mg 100-350 mg. For example, the human dosage ofbupivacaine can be approximately 300 mg.

Methods of Preparation

Some embodiments relate to a process for preparing a large diametersynthetic membrane vesicle(s) composition comprising the steps of,forming a first component by mixing a first aqueous phase and an organicphase, said organic phase comprising an organic solvent, at least oneamphipathic lipid, and at least one neutral lipid, encapsulating saidfirst component in a second aqueous phase to provide a second componentusing an atomizing nozzle as disclosed and described herein, said secondcomponent comprising an aqueous phase, removing the organic solvent fromthe second component to form a composition of large diameter syntheticmembrane vesicle particles, wherein the removing can be accomplished bycontacting the second component with a gas, optionally heating andoptionally filtering the composition by particle concentration. Suchsteps may be combined with other steps. In some embodiments the lipidphase can include cholesterol. In some embodiments, the organic solventcan be a volatile water-immiscible or sparingly miscible solvent. Insome embodiments, the first component can be a first emulsion. In someembodiments, the second component can be a second emulsion. In someembodiments, the second component can be a droplet. In some embodiments,the large diameter synthetic membrane vesicle(s) can be multivesicularliposomes.

Some embodiments relate to a process for preparing a large diametersynthetic membrane vesicle(s) composition comprising the steps of,forming a first component by mixing a first aqueous phase and an organicphase, said organic phase comprising an organic solvent, at least oneamphipathic lipid, and at least one neutral lipid, encapsulating saidfirst component in a second aqueous phase to provide a second component,said second component comprising an aqueous phase, removing the organicsolvent from the second component to form a composition of largediameter synthetic membrane vesicle particles, wherein the removing canbe accomplished by using a solvent removal chamber as disclosed anddescribed herein, and optionally filtering the composition by particleconcentration. Such steps may be combined with other steps. In someembodiments the lipid phase can include cholesterol. In someembodiments, the organic solvent can be a volatile water-immiscible orsparingly miscible solvent. In some embodiments, the first component canbe a first emulsion. In some embodiments, the second component can be asecond emulsion. In some embodiments, the second component can be adroplet. In some embodiments, the large diameter synthetic membranevesicle(s) can be multivesicular liposomes.

Some embodiments relate to a process for preparing a large diametersynthetic membrane vesicle(s) composition comprising the steps of,forming a first component by mixing a first aqueous phase and an organicphase, said organic phase comprising an organic solvent, at least oneamphipathic lipid, and at least one neutral lipid, encapsulating saidfirst component in a second aqueous phase to provide a second componentusing an atomizing nozzle as disclosed and described herein, said secondcomponent comprising an aqueous phase, removing the organic solvent fromthe second component to form a composition of large diameter syntheticmembrane vesicle particles, wherein the removing can be accomplished byusing a solvent removal chamber as disclosed and described herein, andoptionally filtering the composition by particle concentration. Suchsteps may be combined with other steps. In some embodiments the lipidphase can include cholesterol. In some embodiments, the organic solventcan be a volatile water-immiscible or sparingly miscible solvent. Insome embodiments, the first component can be a first emulsion. In someembodiments, the second component can be a second emulsion. In someembodiments, the second component can be a droplet. In some embodiments,the large diameter synthetic membrane vesicle(s) can be multivesicularliposomes.

Some embodiments relate to a process for preparing a multivesicularliposome composition comprising the steps of, forming a first componentby mixing a first aqueous phase and an organic phase, said organic phasecomprising a volatile water-immiscible or sparingly miscible solvent, atleast one amphipathic lipid, and at least one neutral lipid,encapsulating said first component in a second aqueous phase to providea second component, said second component comprising an aqueous phase,removing the volatile water-immiscible or sparingly miscible solventfrom the second component to form a composition of MVL particles,wherein the removing can be accomplished by contacting the secondcomponent with a gas, and optionally filtering the multivesicularliposome composition by particle concentration. Such steps may becombined with other steps. In some embodiments the lipid phase caninclude cholesterol.

First Component

In embodiments that include a first component, the first component canbe formed by mixing two phases, such as an organic phase and a firstaqueous phase. In some embodiments, a therapeutic agent can be added tothe organic phase. In some embodiments, a therapeutic agent can be addedto the first aqueous phase. In some embodiments, a therapeutic agent canbe added to both the organic phase and the first aqueous phase. In someembodiments, the organic phase can include at least one amphipathiclipid, at least one neutral lipid, and an organic solvent. In someembodiments, the therapeutic agent can be in the form of apharmaceutically acceptable salt.

In some embodiments, the mixing of the two phases can be accomplishedusing ultrasound. In some embodiments, the mixing of the two phases canbe accomplished using high pressure emulsification. Such emulsificationutilizes an atomizing nozzle as disclosed and described herein. In someembodiments, the mixing of the two phases can be accomplished usingmechanical processes including using high-shear type devices,rotor/stator and homogenizers, shear-type mixer, static mixer, impeller,porous pipe, any of the disclosed mechanical processes optionally incombination with a heat exchanger, or other processes known to producewater-in-oil emulsions. In some embodiments, the mixing of the twophases can be accomplished using a combination of ultrasound and highpressure emulsification. In some embodiments, the mixing of the twophases can be accomplished using a combination of mechanical processesperformed by a device selected from the group consisting of high-sheartype devices, rotor/stator mixers and homogenizers, shear-type mixer,static mixer, impeller, porous pipe, high energy vibration, injectioninto a high velocity liquid stream such as in an aspirator, and thelike.

In some embodiments, the first component can comprise particles havingan average diameter in the range from about 0.1 μm to about 100 μm. Forexample, the particles can have an average diameter of at least about0.1 μm, 1 μm, 5 μm, 10 μm, 15 μm, 20 μm, 50 μm, or 100 μm, or a diameterwithin a range defined by any of two of the preceding values. In someembodiments, the particles can have an average diameter in the rangefrom about 0.2 μm to about 100 μm, from about 0.2 μm to about 50 μm,from about 0.5 μm to about 30 μm, from about 0.5 μm to about 10 μm, fromabout 10 μm to about 50 μm, from about 15 μm to about 45 μm, or fromabout 20 μm to about 40 μm. In a typical embodiment, the particles canhave an average diameter in the range of from about 0.5 μm to about 3μm.

In some embodiments, the first component can be formed at a temperaturein the range from about −5° C. to about 99° C., from about 0° C. toabout 60° C., from about 2° C. to about 40° C., from about 4° C. toabout 20° C., from about 5° C. to about 50° C., from about 10° C. toabout 40° C., or from about 15° C. to about 35° C., or from about 10° C.to about 20° C.

In some embodiments, the organic solvent can be selected from the groupconsisting of diethyl ether, tert-butylmethyl ether, tetrahydrofuran,sevoflurane, desflurane, isoflurane, and enflurane. In some embodiments,the organic solvent can be selected from the group consisting ofhalothane, chloroform, and dichloromethane. In some embodiments, theorganic solvent can be selected from the group consisting of ethylacetate, hexane, hexanes, cyclohexane, pentane, cyclopentane, petroleumether, and toluene. In some embodiments, the organic solvent can beselected from the group consisting of freons, chlorofluorocarbons (CFCs)and hydrochlorofluorocarbons (HCFCs) with boiling points above 15° C. Ina typical embodiment, the organic solvent can be selected from the groupconsisting of chloroform, and dichloromethane. For example, the organicsolvent can be dichloromethane.

In some embodiments, the first aqueous phase can be selected from thegroup consisting of water solutions including one or more componentsselected from the group consisting of a therapeutic agent, dextrose,lysine, dextrose/lysine, sodium chloride, hydrochloric acid, phosphoricacid, an osmotic pressure adjusting agent such as a sugar, dextrose,sucrose, trehalose, fructose, sorbitan, glycerol, or manitol, atherapeutic agent solubility enhancer, and pH modifying agents such assodium hydroxide, arginine, histidine, sodium borate, acids, bases,(hydroxymethyl)aminomethane, or Good's buffers. In some embodiments, theconcentration of any one component in the aqueous phase, other than thetherapeutic agent, can be in the range from about 1 μM to about 1 M,from about 1 mM to about 500 mM, from about 10 mM to about 400 mM, fromabout 100 mM to about 300 mM. In a typical embodiment, any onetherapeutic agent component if used in the first aqueous phase can be inthe range from about 1 mM to about 1 M. For example, the therapeuticagent component can be approximately 200 mM. In a typical embodiment,the concentration of any one component in the first aqueous phase can bein the range from about 100 mM to about 300 mM. For example, theconcentration can be 200 mM. In some embodiments, the first aqueousphase can include phosphoric acid as a component. In some embodiments,the concentration of phosphoric acid can be in the range from about 1 μMto about 1 M, from about 10 μM to about 750 mM, from about 1 mM to about500 mM or from about 10 mM to about 250 mM. In a typical embodiment, theconcentration of phosphoric acid can be in the range from about 100 mMto about 300 mM. For example, the concentration of phosphoric acid canbe 200 mM.

In some embodiments, the organic phase can include an amphipathic lipid.In some embodiments, the amphipathic lipid can be selected from thegroup consisting of soya lecithin,1,2-dioleoyl-sn-glycero-3-phosphocholine,1,2-dilauroyl-sn-glycero-3-phosphocholine,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-diarachidoyl-sn-glycero-3-phosphocholine,1,2-dibehenoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine,1,2-dieicosenoyl-sn-glycero-3-phosphocholine,1,2-dierucoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol,1,2-dioleoyl-sn-glycero-3-phosphoglycerol, and mixture thereof. In atypical embodiment, the amphipathic lipid can be selected from the groupconsisting of 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC),1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) and mixturesthereof. For example, the amphipathic lipid can be a mixture of1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC) and1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG). In some embodimentsthe ratio of 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC) to1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) can be in the rangefrom about 100:1 to about 1:10, from about 50:1 to about 1:1, from about25:1 to about 2:1, from about 15:1 to about 10:1, from about 10:1 toabout 30:1, or from about 15:1 to about 20:1. For example, the ratio of1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC) to1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) can be about 16.8:1.

In some embodiments, the organic phase can include a neutral lipid. Insome embodiments, the neutral lipid can be selected from the groupconsisting of triolein, tripalmitolein, trimyristolein trilinolein,tributyrin, tricaprylin, tricaproin, and tricaprin, and mixture thereof.In a typical embodiment, the neutral lipid can be selected from thegroup consisting of tricaprylin, tricaproin, and tricaprin, and mixturesthereof. For example, the neutral lipid can be tricaprylin. In someembodiments the ratio of amphipathic lipid to neutral lipid can be inthe range from about 50:1 to about 1:5, from about 25:1 to about 1:2,from about 15:1 to about 1:1, from about 10:1 to about 2:1 or from about6:1 to about 3:1. For example, the ratio of amphipathic lipid to neutrallipid can be about 4.4:1.

In some embodiments, the organic phase can include cholesterol. In someembodiments the ratio of amphipathic lipid to cholesterol can be in therange from about 50:1 to about 1:10, from about 25:1 to about 1:5, fromabout 10:1 to about 1:2, from about 5:1 to about 1:1 or from about 3:1to about 1.5:1. For example, the ratio of amphipathic lipid tocholesterol can be about 1.8:1. In some embodiments the ratio ofcholesterol to neutral lipid can be in the range from about 50:1 toabout 1:10, from about 25:1 to about 1:5, from about 10:1 to about 1:2,from about 5:1 to about 1:1 or from about 3:1 to about 2:1. For example,the ratio of cholesterol to neutral lipid can be about 2.4:1. In atypical embodiment, any one therapeutic agent component if used in theorganic phase can be in the range from about 1 mM to about 1 M. Forexample, the therapeutic agent component can be approximately 200 mM.

In some embodiments, the aqueous phase or organic phase can comprise thetherapeutic agent. In a typical embodiment, the therapeutic agent can beselected from the group including semisynthetic aminoglycosideantibiotics such as amikacin; antidiabetics; peptides such as insulin;antitumor drugs such as paclitaxel; antineoplastics includingcytarabine, 5-fluorouracil and floxuridine; alkaloid opiate analgesicsincluding morphine and hydromorphine; local anesthetics includingbupivacaine; synthetic anti-inflamniatory adrenocortical steroidsincluding dexamethasone; antimetabolites including methotrexate;glycopeptide antibiotics including bleomycin; vincaleukoblastines andstathmokinetic oncolytic agents including vincristine and vinblastine;hormones, plasma proteins, cytokines, growth factors, DNA and RNA from avariety of organisms, and antisense oligonucleotides. In someembodiments, the therapeutic agent can be an amide anesthetic. In someembodiments, the therapeutic agent can be selected from the groupconsisting of bupivacaine, mepivacaine, ropivacaine, lidocaine,pyrrocaine, prilocaine, their stereoisomers, and combinations thereof.In a typical embodiment, the therapeutic agent can be bupivacaine or atherapeutically acceptable salt thereof. For example, bupivacaine can bea free base. In some embodiments, the aqueous phase can comprise an acidin sufficient quantity to maintain the bupivacaine or a therapeuticallyor pharmaceutically acceptable salt thereof in the first aqueous phase.

In some embodiments, the first component can be formed by mixing twophases. In some embodiments, the first component can be an emulsion. Insome embodiments, the first component can be in the form of droplets. Insome embodiments, the first component droplets can be formed by mixingtwo phases, such as an organic phase and a first aqueous phase where thespeed of mixing can control the size of the first component droplets. Insome embodiments, the size of the first component droplets can have anaverage diameter of at least about 0.1 μm, 1 μm, 5 μm, 10 μm, 15 μm, 20μm, 50 μm, or 1000 μm, or a diameter within a range defined by any oftwo of the preceding values. In some embodiments, the average firstcomponent droplet size can be preferably between 0.5 μm and 2 μm. Forexample, the first component droplet size can be approximately 1 μm. Insome embodiments, the first component droplet can be an emulsiondroplet.

Second Component Droplet

In some embodiments, the first component can then be combined with asecond aqueous phase to provide a second component droplet. The secondcomponent droplet can be formed by combining the first component withthe second aqueous phase using a three-fluid nozzle to form a firstcomponent/second aqueous phase mixture. In some embodiments, the firstfluid applied to the nozzle is a first liquid, made up of a firstcomponent, the second fluid applied to the nozzle is a second liquidmade up of a second aqueous phase, and the third fluid can be consideredto be a gas (or a gas/vapor mixture), such as nitrogen gas (or anitrogen gas/aqueous vapor mixture) or air scrubbed of CO₂, or air freeof, or substantially free of CO₂. In some embodiments, contacting thefirst component/second aqueous phase mixture with the third fluidcreates the second component droplets as the gas acts to shear the firstemulstion/second aqueous phase mixture into droplets. In someembodiments, the volume:volume ratio of first component to secondaqueous phase can be in the range of from about 1:1000 to about 1000:1,in the range of from about 1:500 to about 500:1, in the range of fromabout 1:50 to about 50:1, in the range of from about 1:10 to about 5:1,or in the range of from about 1:5 to about 5:1. In a typical embodiment,the volume:volume ratio of first component to second aqueous phase canbe in the range of from about 1:3 to about 3:1. Alternately, thevolume:volume ratio of first component to second aqueous phase can be inthe range of from about 2:1 to 1:2. For example, the volume:volume ratioof first component to second aqueous phase can be about 1:1. In someembodiments, the first component can be an emulsion.

In some embodiments, the second aqueous phase can be selected from thegroup consisting of water solutions of, a therapeutic agent, orpharmaceutically acceptable salt thereof, dextrose, lysine,dextrose/lysine, sodium chloride, hydrochloric acid, phosphoric acid, anosmotic pressure adjusting agent such as a sugar, dextrose, sucrose,trehalose, fructose, sorbitan, glycerol, or manitol, a therapeutic agentsolubility enhancer, pH modifying agents such as sodium hydroxide,arginine, histidine, sodium borate, acids, bases,(hydroxymethyl)aminomethane, or Good's buffers, and mixtures thereof. Insome embodiments, the concentration of any one component in the secondaqueous phase can be in the range from about 1 μM to about 1 M, fromabout 10 μM to about 750 mM, from about 100 μM to about 500 mM, 1 mM toabout 250 mM, from about 10 mM to about 150 mM, from about 50 mM toabout 125 mM, from about 100 mM to about 200 mM, from about 100 mM toabout 500 mM, or from about 200 mM to about 400 mM. In a typicalembodiment, the concentration of any one component in the second aqueousphase can be in the range from about 50 mM to about 270 mM.

In some embodiments, the second aqueous phase can be an aqueousdextrose/lysine solution. In some embodiments, the concentration ofdextrose can be in the range from about 1 mM to about 500 mM from about10 mM to about 300 mM, from about 25 mM to about 200 mM or from about 50mM to about 150 mM. In a typical embodiment, the concentration ofdextrose can be in the range from about 60 mM to about 90 mM. Forexample, the concentration of dextrose can be about 80 mM. In someembodiments, the concentration of lysine can be in the range from about1 mM to about 750 mM, from about 10 mM to about 500 mM, from about 50 mMto about 400 mM or from about 100 mM to about 200 mM. In a typicalembodiment, the concentration of lysine can be in the range from about150 mM to about 250 mM. For example, the concentration of lysine can beabout 200 mM.

In some embodiments, a first component can be mixed with a secondorganic phase comprising one or more second amphipathic lipids prior tocombination with a second aqueous phase to provide a second component.In some embodiments, the second component can be in the form ofdroplets. For example, the second organic phase can include the same ordifferent amphipathic lipids than in the first component. The secondcomponent droplet can be formed by combining the first component withthe second aqueous phase using a three fluid nozzle with an additionalinlet for the second organic phase. The second organic phase canalternatively be pumped into the conduit feeding first component to thethree fluid nozzle with an optional static mixer in the conduit betweenthe point of second organic phase addition and the three fluid nozzle.Some active agents can interact with the lipids (e.g. peptides orproteins, have seen this already, especially the charged lipids like PG,e.g. DPPG). For this and other reasons e.g. biocompatibility, storagestability, vibration stability or release rate modification, it may bedesirable to have the outside layer of phospholipids compositiondifferent from the inside chamber wall composition (e.g. PG only on theoutside where it gives charge stability to the MVL but not presentduring high shear mixing with the active agent first aqueous). In someembodiments, the outside layer of phospholipid is put on the particlewhen the first component contacts the second aqueous phase, inside thenozzle and is subsequently atomized and it comes from the phospholipidsleft dissolved in the solvent after the first emulsion is made.Phospholipids with the desired outside composition, dissolved insolvent, can be injected into the first fluid conduit, just before the 3fluid nozzle, and just before an optional static or other mixer so thatthese lipids are present and dissolved in the solvent phase of the firstcomponent. These phospholipids would only contact active containingfirst component droplets for seconds under mild shear condition andwould thus not interact appreciably with the outer layer lipids. Some ofthese added lipids may get incorporated between the chambers of theforming MVL which the 4 fluid nozzle avoids.

In some embodiments, the one or more second amphipathic lipids in thesecond organic phase can be selected from the group consisting of soyalecithin, 1,2-dioleoyl-sn-glycero-3-phosphocholine,1,2-dilauroyl-sn-glycero-3-phosphocholine,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-diarachidoyl-sn-glycero-3-phosphocholine,1,2-dibehenoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine,1,2-dieicosenoyl-sn-glycero-3-phosphocholine,1,2-dierucoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol,1,2-dioleoyl-sn-glycero-3-phosphoglycerol, and mixture thereof. In atypical embodiment, the amphipathic lipid can be selected from the groupconsisting of 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC),1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) and mixturesthereof.

Solvent Removal

In some embodiments, the organic solvent, such as methylene chloride,can be nearly or completely removed from a droplet by further contactingthe droplet with a gas (or a gas with some degree of aqueous phasevapor), such as nitrogen gas, or air scrubbed of CO2, or airsubstantially free of CO2 in a solvent removal chamber. As describedbelow, in some embodiments, the gas can have 50% to 100% relativehumidity. For example, the humidity can be 100%. In some embodiments,the droplet can remain suspended in the gas within the solvent removalchamber until nearly all of the organic solvent is removed therebycreating a large diameter synthetic membrane vesicle particle. In someembodiments, the solvent removal can be done in a solvent removalchamber as discussed herein. The large diameter synthetic membranevesicle particle can then be collected along with multiple other largediameter synthetic membrane vesicle particles, forming large diametersynthetic membrane vesicles suspended in a continuous aqueous phase. Insome embodiments, the large diameter synthetic membrane vesiclessuspended in a continuous aqueous phase can undergo further processing.In some embodiments, the large diameter synthetic membrane vesicles canbe multivesicular liposomes.

Particle Concentration

Optionally, the large diameter synthetic membrane vesicles suspended inthe continuous aqueous solution can be processed to modify/exchange thecontinuous aqueous solution via a particle concentration system. In thisdocument the term concentration unit, concentration apparatus,concentration system, particle-concentration system,particle-concentrating device, and particle concentrator are meant toencompass units and processes that remove some of the particlesuspending medium of a particle suspension and therefore concentratingthe particle concentration as well as encompassing the exchange of thesuspending medium with a new suspending medium, performed in one step orincrementally. These two processes are closely related as exchanging thesuspending medium can be accomplished by concentrating the suspensionand adding new suspending medium. These terms relate to concentratingthe particle suspension and exchanging the suspending medium to be doneseparately or simultaneously. In some embodiments, the large diametersynthetic membrane vesicles can be multivesicular liposomes.

In one embodiment, the continuous aqueous solution can bemodified/exchanged by diafiltration or cross-flow filtration. Thediafiltration can achieve several objectives, including: exchanging anaqueous solution by an isotonic solution, concentrating of the largediameter synthetic membrane vesicles particles, removing unencapsulateddrug, and removing of residual organic solvent. In some embodiments, thelarge diameter synthetic membrane vesicles can be multivesicularliposomes.

Particle concentration, such as through diafiltration, can be a methodemployed for the filtration, purification, and separation of the largediameter synthetic membrane vesicles particles from complex mixtures byvirtue of the physical characteristics of the large diameter syntheticmembrane vesicles particles. For example, the most common physicalcharacteristic used is size. This filtration can involve cross-flowfiltration, as opposed to dead-end filtration. In cross-flow filtration,a suspension can be circulated under pressure and in contact with afilter, so that a permeate (the material which passes through thefilter) leaves the system, and a retentate (the material which does notpass through the filter) is left behind and exits the filter housingthrough a port different than the permeate and can be recirculatedthrough the filter. The suspension then becomes concentrated in materialthat does not pass through the filter. In processes in which a firstsolution is to be exchanged for a second solution, the second solutionis introduced on the retentate side of the filter, until the permeategradually consists of the second solution. By this time, the firstsolution has been flushed from the suspension. An additional consequenceof suspending medium exchange is to further remove any residual organicsolvent, such as methylene chloride which has a small but appreciablewater solubility and is thus removed in the permeate stream. Likewisethe therapeutic agent released during the large diameter syntheticmembrane vesicles formation can also be removed in the permeate. In someembodiments, the large diameter synthetic membrane vesicles can bemultivesicular liposomes.

In some embodiments, aseptic methods can be included with any of thedescribed methods and apparatus described herein.

Although sterilization of a final filled container as a dosage form isoften a preferred process in pharmaceutical manufacture for theassurance of minimal risk of microbial contamination in a lot, the largediameter synthetic membrane vesicles manufactured in the presentprocesses can be susceptible to unacceptable damage when subjected tosome terminal sterilization techniques, such as autoclaving and gammairradiation. In the absence of validated, non-damaging terminalsterilization, some embodiments of the invention utilize aseptictechniques, in which the product is prepared according to a carefullydesigned series of aseptic steps. These are designed to prevent theintroduction of viable microorganisms into components, where sterile, oronce an intermediate process has rendered the bulk product or itscomponents free from viable microorganisms. Products defined asaseptically processed can consist of components that have beensterilized by aseptic means. For example, bulk products which arefilterable liquids, can be sterilized by filtration. Final emptycontainer components can be sterilized by heat; dry heat for glass vialsand autoclaving for rubber seal components. The requirements forproperly designed, validated and maintained filling and processingfacilities are directed to: an air environment free from viablemicroorganisms, and designed to permit effective maintenance of airsupply units; training of personnel who are adequately equipped andgowned. Available published standards for controlled work areas include:Federal Standard No. 209B, Clean Room and Work Station Requirements fora Controlled Environment, Apr. 24, 1973; NASA Standard for Clean Roomand Work Stations for Microbially Controlled Environment, publicationNHB5340.2, August 1967; and Contamination Control of AerospaceFacilities, U.S. Air Force, T. O. 00-25-203, Dec. 1, 1972, change 1,Oct. 1, 1974. In some embodiments, the large diameter synthetic membranevesicles can be multivesicular liposomes.

In aseptic processing, one of the most important laboratory controls isthe establishment of an environmental monitoring program. Samples can becollected from areas in which components and product are exposed to theenvironment, including mixing rooms and component preparation areas.Microbiological quality of aseptic processing areas is monitored todetermine whether or not aseptic conditions are maintained duringfilling and closing activities. Routine sampling and testing of the roomair, floors, walls, and equipment surfaces is carried out. This programestablishes the effectiveness of cleaning and sanitizing equipment andproduct contact surfaces, and ensures that potential contaminants areheld to an acceptable level. The disinfectants are checked to assurethat their efficacy against normal microbial flora is maintained.Sampling schedules, including locations and frequency of sampling, aremaintained. Passive air samplers such as settling plates (Petri dishes)are employed as well.

Aseptic assembly operations can be validated by the use of amicrobiological growth nutrient medium to simulate sterile productfilling operations, known as “sterile media fills.” The nutrient mediumcan be manipulated and exposed to the operators, equipment, surfaces andenvironmental conditions to closely simulate the same exposure which theproduct itself will undergo. The sealed drug product containers filledwith the media can then be incubated to detect microbiological growthand the results are assessed to determine the probability that any givenunit of drug product may become contaminated during actual filling andclosing operations. Media filling, in conjunction with comprehensiveenvironmental monitoring can be particularly valuable in validating theaseptic processing of sterile solutions, suspensions, and powders.Filling liquid media, as part of validating the processing of powders,may necessitate use of equipment and/or processing steps that wouldotherwise not be attendant to routine powder operations.

In some embodiments, clean-in-place (CIP) and sterilize-in-place (SIP)procedures can be utilized by methods generally known in the art. Someembodiments include monitoring of the temperature at the steam traps.According to this procedure, as steam is admitted into the vessels andfill lines to effect sterilization, the temperature at the outlet pointsis monitored until bacterial kill is assured. At this point, the sealscan be closed, and the system can be sterilized for further use.Subsequently, the equipment can be used aseptically in a non-sterileroom environment. The systems described herein, may also compriseadditional components (e.g. valves, steam lines, condensate drains) tofacilitate sterilization by steaming-in-place.

Sterility testing of product lots can be carried out directly after thelot is manufactured as a final product quality control test. Testing canbe done in accordance with various procedures found in the U.S.Pharmacopeia (U.S. P.) and FDA regulations.

Sterile filtration of all fluids which enter the manufacturing system isessential for an aseptic process, as envisioned in certain embodimentsof the present application. Rating of pore sizes of filter membranes isby a nominal rating reflecting the capacity of the membrane to retainmicroorganisms of size represented by specified trains, not bydetermination of an average pore size and statement of distribution ofsizes. Sterilizing filter membranes include those capable of retaining100% of a culture of 10⁷ organisms of a strain of Pseudomonas diminua(ATTC 19146) per square cm of membrane surface under a pressure of notless than 30 psi. Such filter membranes can be nominally rated 0.22 μmor 0.2 μm, depending on the manufacturer. Bacterial filter membranescapable of retaining only larger microorganisms (including SerratiaMarcescens (ATTC 14756)) are labeled with a nominal rating of 0.45 μm.Filter membranes used in the present processes can be of the 0.2 μmtype, and can be used in all lines feeding from liquid solution and gasstorage tanks to vessels and transfer lines used in the methodsdisclosed herein.

In some embodiments the process apparatus is sterilized before use andisolated from the environment by the use of sterilizing filters on allapparatus inputs and outputs where the apparatus includes the sterilizedproduct vessel. In this embodiment sterile product can be produced in anaseptic fashion, in a non sterile environment.

DESCRIPTION OF THE INSTANT DEVICES AND THEIR METHODS OF USE

The present embodiments will now be described in more detail in terms offeatures and operations with reference to the accompanying drawings.

FIG. 1A

An embodiment of the present application is a continuous-flow system formanufacturing pharmaceutical formulations. An example of such acontinuous-flow system is presented in FIG. 1A. FIG. 1A is a schematicdiagram of the significant components and sub-systems used in a systemfor manufacturing pharmaceutical formulations. Each of the componentsand sub-systems can be included in, and operated as, a part of thelarger manufacturing system or may, in the alternative, be runautonomously in a continuous fashion to accomplish the objective of eachsub-system as described herein. Additionally, each sub-system may be runin a continuous-flow manner using batch inputs and producing a batchoutput.

In one embodiment, the manufacturing system 100 is comprised of a tank5, which can hold a first fluid, the first fluid can be pumped by apositive-displacement pump 2 through a hydrophilic sterilizing filter 15and into a high shear mixer 25. In some embodiments, the first fluid canbe an aqueous solution. In some embodiments, the first fluid can be afirst liquid. In some embodiments, the first liquid can be an aqueoussolution. Similarly, tank 10 can feed a second fluid, via apositive-displacement pump 12, through a hydrophobic sterilizing filter20 into the high shear mixer 25. In some embodiments, the second fluidcan be a second liquid. In some embodiments, the second liquid can be anorganic solvent. The high shear mixer 25, a heat exchanger 30, andassociated inlet line 96 and outlet line 97 comprise one embodiment of afirst sub-system. In some embodiments, the first sub-system can be afirst emulsification sub-system. In some embodiments, the high shearmixer 25 can create a first dispersion of aqueous particles (the“discontinuous phase”) suspended in an organic continuous phase. In someembodiments, the first dispersion can be a first component. In someembodiments, a majority of the first component can then be fed from thehigh shear mixer 25 into the heat exchanger 30, and the cooled firstcomponent can flow back to the high shear mixer 25. In one embodiment,however, a portion of the emulsion leaving the high shear mixer 25 canbe forced towards a nozzle 75, for example by the addition of additionalorganic solvent and aqueous solution to the first emulsificationsub-system.

In some embodiments, the emulsion leaving the high shear mixer 25 can betransferred to an evaporation apparatus or evaporation sub-system. Inone embodiment the evaporation apparatus, or evaporation sub-system, iscomprised of a solvent removal vessel 50, the atomizing nozzle 75, a gasinlet 115, and a gas outlet 80. In some embodiments, the nozzle 75 actsto spray atomized droplets of the first component into the solventremoval vessel 50. In some embodiments, the nozzle 75 can combine thefirst component with a fluid from a tank 60 and spray the firstcomponent/liquid mixture into the solvent removal vessel 50 as atomizeddroplets. In some embodiments, the fluid from the tank 60 is a liquid.In some embodiments, the liquid from the tank 60 is a buffer solution.In some embodiments, the buffer solution can be fed by apositive-displacement pump 22 from the tank 60 through a hydrophilicsterile filter 65 into the nozzle 75. Gas from the gas supply can bepassed through a pressure regulator 11 and a sterilizing gas filtrationsystem 35 before entering the nozzle 75. In some embodiments, the gasacts to atomize the first component/liquid mixture as it exits thenozzle 75 into the solvent removal vessel 50.

In some embodiments, a carrier gas can be supplied to the solventremoval vessel 50 from the gas supply and enter the solvent removalvessel through the gas inlet 115. The carrier gas can be, for example,nitrogen gas (or a nitrogen gas/aqueous vapor mixture) or air scrubbedof CO₂. The pressure of the carrier gas can be regulated using apressure regulator 31. In some embodiments, the carrier gas first passesthrough a heater/humidifier 90 and then through a gas filtration system45 before entering the solvent removal vessel 50 through the gas inlet115. In some embodiments, the carrier gas circulates through the solventremoval vessel 50, and creates an intense gas vortex, which canfacilitate solvent evaporation from the atomized droplets as they aresprayed into the solvent removal vessel 50 from the nozzle 75. The gasvortex can substantially prevent the atomized droplets from exiting thesovlent removal chamber with the carrier gas through gas outlet 80. Withevaporation of the solvent, the atomized droplets become droplets oflarge diameter synthetic membrane vesicles within an aqueous phase. Insome embodiments, the atomized droplets can be first component/liquidmixture droplets. In some embodiments, the large diameter syntheticmembrane vesicles can be within an aqueous phase. In some embodiments,the large diameter synthetic membrane vesicles can be multivesicularliposomes. In some embodiments, the carrier gas and evaporated solventcan then be removed from the solvent removal vessel 50 through the gasoutlet 80, after which they can then pass through a filtration system 55comprised of a pre-filter and a sterilizing barrier filter, before beingremoved as waste 95. In some embodiments, the prefilter (not shown) canbe a high efficiency cyclone separator.

In one embodiment, the evaporation sub-system is comprised of aplurality of solvent removal vessels, each with one or more atomizingnozzles, used in parallel to evaporate the solvent from the atomizeddroplets. In some embodiments, the evaporation sub-system can becomprised of a plurality of solvent removal vessels, such as solventremoval vessel 50, each with one or more atomizing nozzles, such asatomizing nozzle 75, used in parallel to evaporate solvent from theatomized droplets. In some embodiments, the solvent removal vessel canhave additional multiple atomizing nozzles (not shown) such as 75.

In some embodiments, a portion of the gas passing through theheater/humidifier 90 can be directed towards a gas inlet 110 located inthe lid of the solvent removal vessel 50. In some embodiments, the gasinlet 110 allows gas to enter the solvent removal vessel 50 andcirculate in the upper portion of the vessel acting to prevent particlebuildup on the lid.

In some embodiments, a two-fluid rinse nozzle 105 can be placed in andthrough the lid of the solvent removal vessel 50. In some embodiments,the rinse nozzle 105 can spray the wall of solvent removal vessel 50.The rinse nozzle 105, which receives a buffer solution from a buffersolution tank 66, through a pump 64 and a sterilizing filter 62, canspray atomized tank wall rinse solution particles into the solventremoval vessel 50. The buffer solution can be atomized by gas travelinginto the nozzle 105 through a pressure regulator 21 and a sterilizinggas filtration system 85. Spraying atomized wall rinse solutionparticles into the solvent removal vessel 50 can act to prevent thelarge diameter synthetic membrane vesicles droplets (FIG. 7, component7380) from sticking to the walls of the solvent removal vessel 50 byrinsing or flushing particles from the internal surfaces of the vessel50, and out a drain port 130 on the bottom of the vessel 50.

Removal of the solvent from the atomized droplets, affords largediameter synthetic membrane vesicles coated in a buffer solution shellas droplets. In some embodiments, the large diameter synthetic membranevesicles can be multivesicular liposomes. These droplets then cancollect on the bottom of the solvent removal vessel 50 to form asuspension of large diameter synthetic membrane vesicles in a buffersolution. This large diameter synthetic membrane vesicles suspension cansubsequently be pumped by a pump 125 through a solvent removal vesseloutlet line 120 to an optional sub-system 70. In some embodiments, thelarge diameter synthetic membrane vesicles suspension can be pumped by apump 125 through a continuous heat treatment system 150 before enteringthe option sub-system 70. In some embodiments, the buffer solution canbe exchanged for, for example, a saline solution. In some embodiments,the sub-system 70 can be a single or series of concentration unitsrunning in batch or continuous mode. In a typical embodiment, theconcentration units can be run in continuous mode. In a typicalembodiment, large diameter synthetic membrane vesicles can beconcentrated and the resulting suspension collected by the process. Insome embodiments, the large diameter synthetic membrane vesicles can bemultivesicular liposomes.

In some embodiments, one or more components of the manufacturing system100 can be omitted from the system.

FIG. 1B

In some embodiments, the manufacturing system 100 further comprises amass flow controller 13, a mass flow controller 23, a mass flowcontroller 33, a rotometer flow indicator 57, a heater 93, a steamgenerator 40, and a metering pump 6 as shown in FIG. 1B. Each of thecomponents and sub-systems can be included in, and operated as, a partof the larger manufacturing system or may, in the alternative, be runautonomously in a continuous fashion to accomplish the objective of eachsub-system as described herein. Additionally, each sub-system may be runin a continuous-flow manner using batch inputs and producing a batchoutput.

In some embodiments, the mass flow controllers 13, 23, and 33 measure,indicate, and control the gas flow supply to their associated apparatus.In some embodiments, the rotometer flow indicator 57 measures andindicates the gas flow to the lid protection nozzle (FIG. 1A 110 7430.In some embodiments, the steam generator 40 allows precisehumidification of the carrier gas. In some embodiments, the meteringpump 6 allows precise control of the water vapor generated by the steamgenerator 40.

FIG. 1C

One embodiment of the present application includes a continuous heattreatment system. FIG. 1C provides a schematic of one example of acontinuous heat treatment system 150. In some embodiments, the largediameter synthetic membrane vesicles suspension can be pumped from thesolvent removal vessel (FIG. 1A, component 50) flowing through a feedline 120 (also seen in FIG. 1A, component 120), to the continuous heattreatment system 150 before entering a subsystem (FIG. 1A, component70).

In some embodiments, the continuous heat treatment system comprises atemperature controlled tank 151, optionally a temperature control jacket(not shown), a pressurized tank 160, a holding coil tubing 156, and anitrogen source 157 as shown in FIG. 1C. Each of the components andsub-systems can be included in, and operated as, a part of the largermanufacturing system or may, in the alternative, be run autonomously ina continuous fashion to accomplish the objective of each sub-system asdescribed herein. Additionally, each sub-system may be run in acontinuous-flow manner using batch inputs and producing a batch output.

In some embodiments, a portion of the large diameter synthetic membranevesicles suspension can be pumped by a pump (FIG. 1A, component 125)through the feed line 120 into a mixing vessel 180, for example anin-line static mixer. In some embodiments, the large diameter syntheticmembrane vesicles suspension, flowing through the line 120, can comeinto contact with a solution from the tank 151 before flowing through aline 155 into the mixing vessel 180. In one embodiment, the suspensionfeeding the mixing vessel 180 through the line 120, flows at 165 ml/minand the solution through the line 155 flows at 247.5 ml/min, In someembodiments, the solution can be fed through a line 152 by a pump 153,the solution first passing through a hydrophilic sterile filter 154 at arate of 1.5 L per 1 L of the large diameter synthetic membrane vesiclessuspension added to the mixing vessel 180. In some embodiments, the tank151 is temperature controlled. In some embodiments, the solution fromthe tank 151 is a dextrose solution. In some embodiments, the dextrosesolution is heated to about 98° C. in the temperature controlled tank151. In some embodiments, the suspension/dextrose mixture flows throughthe holding coil tubing 156. In some embodiments, the holding coiltubing 156 holds the suspension/dextrose mixture for a specifiedtreatment time. In some embodiments, the treatment time is between 10seconds and 30 seconds. In some embodiments, the suspension/dextrosemixture is continuously heated for 30 seconds to a temperature at orabove 60 C. In some embodiments, the holding coil tubing 156 has avolume of 206 ml.

In some embodiments, the suspension/dextrose mixture can leave theholding coil tubing 156 to enter a retentate vessel 168. In someembodiments, a solution can be fed from the tank 160 into the retentatevessel 168 through a line 164, the solution first passing through amanual valve 162 and a sterilizing hydrophilic filter 163, at a rate of1.4 L per 1 L suspension/dextrose mixture added to the retentate vessel168. In some embodiments, the solution flows through the line 164 at arate of 578 ml/min. In some embodiments, the tank 160 is temperaturecontrolled. In some embodiments, the temperature controlled tank ispressurized. In some embodiments, the tank 160 is pressurized with thenitrogen source 157 through a line 158 and a manual valve 159. In someembodiments, the solution is a saline solution. In some embodiments, thesaline solution is a cold saline solution. In some embodiments, theretentate vessel 168 is temperature controlled or cooled.

In some embodiments, a portion of the suspension/dextrose mixture in theretentate vessel 168 can be pumped by a pump 171 through a cross-flow(tangential-flow) filtration module 167. In some embodiments, thepermeate can be drawn off through a permeate line 173 (passing through asterilizing hydrophilic filter 172 and a manual valve 174), wherein foreach volume of suspension/dextrose mixture added to the retentate vessel168, a volume can be removed and discarded. In some embodiments, theretentate from the filtration module 167 can be circulated back into theretentate vessel 168 via a retentate line 166. In some embodiments, foreach volume of suspension/dextrose mixture added to the retentate vessel168, a volume of the liquid can be removed from the retentate vessel 168through a feed line 169 and a metering pump 170 to be further processedby a subsystem (as seen in FIG. 1A, component 70; systems of FIG. 8,FIG. 10 and FIG. 11). In some embodiments, the suspension can flowthrough the pump 170 at a rate of 490 ml/min.

Filters 154, 163, and 172 are sterilizing hydrophilic filters. Filters,161 and 176 are sterilizing hydrophobic gas vent filters used in thevessels and fed by gas lines 165 and 175, respectively.

FIG. 2

One embodiment of the present application includes an emulsificationsystem, the process of using the system, and the large diametersynthetic membrane vesicles products made by the process. In someembodiments, the large diameter synthetic membrane vesicles can bemultivesicular liposomes. FIG. 2 provides a schematic of one example ofa continuous-flow emulsification system 210. The emulsification system210 includes a high shear mixer 2130 and a heat exchanger 2170. In someembodiments, the high shear mixer 2130 can be used in preparing a firstcomponent. The high shear mixer 2130 used in one embodiment is the RossHSM-703XS-20 Sanitary Inline High Shear Mixer made by the Charles Ross &Son Company of Hauppauge, N.Y. In some embodiments, the head of the highshear mixer 2130 can be connected to an aqueous inlet line 2120. In someembodiments, the head of the high shear mixer 2130 can be fed an aqueoussolution through the aqueous inlet line 2120. In some embodiments, thehead of the high shear mixer 2130 can be connected to a recirculationline 2125. In some embodiments, the high shear mixer 2130 can feed arecirculated emulsion through the recirculation line 2125. Therecirculated emulsion contains already-sheared aqueous dropletsdispersed in a continuous organic solution. In some embodiments, theaqueous solution can be stored in a tank (FIG. 1A, component 10) andpass through a liquid sterilization filter (FIG. 1A, component 20)attached to the aqueous inlet line 2120 before entering the high shearmixer 2130 at various volumetric flow rates. High shear mixers areavailable to deliver volumetric flow rates between 1 mL/minute and 4,000mL/minute, between 10 mL/minute and 1,000 mL/minute, between 10mL/minute and 100 mL/minute, and preferably between 15 mL/minute and 50mL/minute. In some embodiments, the aqueous inlet line 2120 can beconfigured to run co-axially through a portion of the recirculation line2125. The range of volumetric flow rates above is appropriate whencontemplating the use of one, or optionally multiple, atomizing nozzles.

In some embodiments, the aqueous solution can be injected through thecenter of a stator inside of the head of the high shear mixer 2130 intothe center of a spinning rotor (not shown) also inside the high shearmixer head 2130. As the aqueous phase passes between the rotor and thestator, inside the high shear mixer head 2130, it is sheared by therotor teeth and stator teeth into aqueous droplets. The sheared aqueousdroplets become part of a flowing stream of the recirculated emulsionwhich itself can be fed back into the high shear mixer through therecirculation line 2125. The combined aqueous solution and recirculatedemulsion can pass through blade shear gaps (not shown) in the high shearmixer head 2130, as it travels from the center to the outside of therotor. In one embodiment, on average, aqueous droplets are recirculatedapproximately 100 times through the emulsification system 210, andthrough that process, the aqueous droplets are sheared roughly 1,300times by the blade shear gaps (not shown).

Exiting the high shear mixer head 2130 through an exit line 2150 is theresultant emulsion, with aqueous droplets dispersed in the organiccontinuous phase, traveling at a volumetric flow rate between 1 L/minuteand 500 L/minute, between 5 L/minute and 100 L/minute, and preferablybetween 10 L/minute and 50 L/minute. The major portion of the emulsionexiting the high shear mixer 2130, approximately between 50% and 99.99%,between 70% and 99.9%, and preferably between 80% and 99.9%, is passedthrough the heat exchanger 2170 to cool the emulsion which has beenheated by the mechanical shearing process of the high shear mixer 2130.Additionally, a smaller portion of the recirculating emulsion,approximately between 1 mL/minute and 8,000 mL/minute, between 10mL/minute and 2,000 mL/minute, between 20 mL/minute and 200 mL/minute,and preferably between 40 mL/minute and 100 mL/minute, travels from thehigh shear mixer 2130 through a nozzle feed line 2180 to a nozzle foruse in making the first component/buffer atomized droplets. The range ofvolumetric flow rates above is appropriate when contemplating the use ofone, or optionally multiple, atomizing nozzles. In some embodiments, theheat exchanger 2170 can be comprised of a series of coils surrounded bycirculating solution. In some embodiments, the circulating solution canbe at a temperature ranging from about −5° C. to about 30° C. In someembodiments, the circulating solution can be a glycol/water solution. Insome embodiments, the glycol/water solution can be at a temperatureranging from about 0° C. to about 10° C. In some embodiments, the heatexchanger 2170 is comprised of a series of coils surrounded bycirculating 5° C. glycol/water solution. In some embodiments, thecirculating glycol/water solution can be fed to the heat exchanger 2170through an inlet line 2110 and an outlet line 2105. In some embodiments,the emulsion travels through the heat exchanger coils, being cooled bythe glycol/water solution, and exits the heat exchanger through a heatexchanger outlet 2175. In some embodiments, the heat exchanger outlet2175 can carry the cooled emulsion back through the recirculation line2125 for additional mixing with added aqueous phase and added organicsolution. In some embodiments, the high shear mixer 2130 can act as ahigh volume centrifugal pump to drive a high volumetric flow ratethrough the heat exchanger and around the recirculation loop, back tothe high shear mixer head 2130.

Attached to the recirculation line 2125 is an organic solution inletline 2160, which can be used to replenish the organic solution in theemulsification system 210 according to the volumetric amount of organicsolution fed in the first component to the nozzle feed line 2180. Insome embodiments, the organic solution can be stored in a tank (notshown) and flows, at a volumetric flow rate between 1 mL/minute and4,000 mL/minute, between 10 mL/minute and 1,000 mL/minute, between 10mL/minute and 100 mL/minute, and preferably between 20 mL/minute and 50mL/minute. The range of volumetric flow rates above is appropriate whencontemplating the use of one, or optionally multiple, atomizing nozzles.In some embodiments, the organic solution flows through a liquidsterilization filter (shown in FIG. 1A and FIG. 1B, component 15)attached to the organic inlet line 2160 before entering therecirculation line 2125 and the high shear mixer 2130. In oneembodiment, the volumetric flow rate of the organic solution addedthrough the inlet line 2160 can be equal to the volumetric flow rate ofaqueous solution added to the high shear mixer 2130 through the aqueousinlet line 2120. That is, in one embodiment, the volumetric flow ratesof the added liquids are at a 1:1 ratio. At steady-state, because theseare incompressible fluids and the piping described herein isnon-expandable piping, the flow rate of the first component traveling tothe nozzle through the nozzle feed line 2180 is equal to the sum of theflow rates of the organic solution being replenished to theemulsification system 210 through the organic phase inlet line 2160 andthe aqueous solution being replenished to the emulsification system 210through the aqueous inlet line 2120.

In one embodiment, the emulsification system is used autonomously tomake MVL where the first component is collected in a container andfurther processed to make the second emulsion. The second emulsion isthen sparged and filtered in a batch mode as described in PCT PatentPublication No. WO99/13865 to S. Kim et al., incorporated herein byreference.

In one embodiment, the emulsification system is employed autonomously toproduce common emulsions of triglycerides (vegetable oils) andsurfactants (Tween-20, Pluronic F-66, egg lecithin) and water withcommon additives (scents, flavors). In an additional embodiment, acosmetic lotion is made with a triglyceride as the discontinuous phase(oil in water emulsion). In still other embodiments, the emulsificationsystem produces ointments, creams and salves, with the triglyceride asthe continuous phase. Additionally, a triglyceride emulsion in waterwith egg yolk as the surfactant is used in the emulsification system toproduce a type of edible mayonnaise.

FIG. 3A-6

One embodiment of the present application includes an atomizing nozzle,the process of using the nozzle, and the large diameter syntheticmembrane vesicles made by the process. In some embodiments, the largediameter synthetic membrane vesicles can be multivesicular liposomes.Examples of the instant atomizing nozzles, the processes for using them,as well as the MVL products, are presented in FIGS. 3A-6 and describedherein.

FIG. 3A

FIG. 3A is a schematic partial view of an atomizing nozzle 310,providing a cross-sectional view of the lower portion of the atomizingnozzle including a fluid contacting chamber 3125. The fluid contactingchamber 3125 is conically tapered from the bottom of an inner fluidconduit 3165 to a cylindrical tip 3145. The fluid contacting chamber3125 is annularly surrounded by an annular gas chamber (a third fluidconduit) 3135, which narrows to form an annular gas orifice 3150. Thebottom portion of the cylindrical tip 3145 is annularly andconcentrically surrounded by the annular gas orifice 3150.

In some embodiments, a first fluid 3115 can travel at a volumetric flowrate between 2 mL/minute and 1,000 mL/minute, between 10 mL/minute and500 mL/minute, between 20 mL/minute and 100 mL/minute, and preferablybetween 50 mL/minute and 100 mL/minute through an inner fluid conduit(central needle) 3165. In a typical embodiment, the first fluid 3115 isan emulsion. In some embodiments, the first fluid exits the bottom 3163of the inner fluid conduit 3165 and enters the fluid contacting chamber3125, whereupon the first fluid 3115 comes in physical communicationwith a second fluid 3120. In a typical embodiment, the second fluid 3120is a buffer solution. In some embodiments, the second fluid 3120 travelsat a volumetric flow rate between 2 mL/minute and 1,000 mL/minute,between 10 mL/minute and 500 mL/minute, between 20 mL/minute and 100mL/minute, and preferably between 50 mL/minute and 100 mL/minute,through the outer fluid conduit 3123, which annularly surrounds theinner fluid conduit 3165 (as seen from the downward perspective view Band displayed in FIG. 3B), to reach the fluid contacting chamber 3125.The above atomizing nozzle flow rates are for a single atomizing nozzle.In some embodiments, the first fluid 3115 forms a cylindrical coretraveling through the fluid contacting chamber 3125, now being inphysical communication with, and annularly surrounded by, the secondfluid 3120. In some embodiments, the first fluid and the second fluidare immiscible. In some embodiments, the first fluid and second fluidcan be sparingly miscible. In some embodiments, the immiscibilitybetween the second fluid 3120 and the first fluid 3115, and the velocityat which the second fluid 3120 and the first fluid 3115, causes thesecond fluid 3120 to form a sheath around the first fluid 3115 core. Ina typical embodiment, the first fluid 3115 is an emulsion and the secondfluid 3120 is a buffer solution.

In some embodiments, the diameter of the fluid contacting chamber 3125can be conically narrowed as it joins the cylindrical tip 3145. As thefirst fluid 3115 travels through the narrowing fluid contacting chamber3125 and into the cylindrical tip 3145, the diameter of the first fluid3115 core is correspondingly decreased. Likewise, the second fluid 3120can be constricted to create a thinner concentric annular sheath aroundthe first fluid 3115 core. As a result of decreasing the diameter of thefluid contacting chamber 3125 and passing the fluids through thecylindrical tip 3145, the velocities of the first fluid 3115 and secondfluid 3120 are increased. In a typical embodiment, the first fluid 3115is an emulsion and the second fluid 3120 is a buffer solution.

FIG. 3C shows an expanded cross-sectional view of the first fluid andsecond fluid in the cylindrical tip 3145. In some embodiments, the ratioof the diameters of the first fluid to the second fluid in the nozzletip can be the same as the ratio of the diameters of the first fluid ininner fluid conduit 3165 to the second fluid in the outer fluid conduit3123. In some embodiments, the ratio of the diameters of the first fluidto the second fluid in the nozzle tip can be larger than the ratio ofthe diameters of the first fluid in the inner fluid conduit 3165 to thesecond fluid in the outer fluid conduit 3123. In some embodiments, theratio of the diameters of the first fluid to the second fluid in thenozzle tip can be smaller than the ratio of the diameters of the firstfluid in inner fluid conduit 3165 to the second fluid in the outer fluidconduit 3123.

FIG. 3D shows an expanded cross-sectional view of the first fluid andsecond fluid in a droplet 3155. In some embodiments, the ratio of thediameters of the first fluid to the second fluid in a droplet 3155 canbe the same as the ratio of the diameters of the first fluid in theinner fluid conduit 3165 to the second fluid in the outer fluid conduit3123. In some embodiments, the ratio of the diameters of the first fluidto the second fluid in a droplet 3155 can be larger than the ratio ofthe diameters of the first fluid in the inner fluid conduit 3165 to thesecond fluid in the outer fluid conduit 3123. In some embodiments, theratio of the diameters of the first fluid to the second fluid in adroplet 3155 can be smaller than the ratio of the diameters of the firstfluid in the inner fluid conduit 3165 to the second fluid in the outerfluid conduit 3123.

The nozzle 310 comprises a gas input channel 3130 which supplies a gas3140. In some embodiments, the volumetric flow rate of the gas can bebetween 30 L/minute and 1,000 L/minute, between 30 L/minute and 500L/minute, between 20 L/minute and 200 L/minute, and preferably between25 L/minute and 100 L/minute and at a pressure between 5 psig and 1,000psig, between 10 psig and 250 psig, between 20 psig and 150 psig, andpreferably between 40 psig and 120 psig to an annular gas chamber 3135.The above atomizing nozzle flow rates are for a single atomizing nozzle.Prior to reaching nozzle 310, the gas 3140 can be passed through a gasfiltration system (not shown) comprising a hydrophobic gas sterilizer(as seen in FIG. 1A and FIG. 1b , component 35). The wall of the fluidcontacting chamber 3125 provides a barrier between the annular gaschamber 3135 and the fluid contacting chamber 3125 such that there is nophysical contact between the fluids in the fluid contacting chamber 3125and the gas 3140 in the annular gas chamber 3135. In some embodiments,the outlet of the cylindrical tip 3145 and the outlet of the annular gasorifice 3150 can be flush with each other such that second fluid 3120does not come into contact with the gas 3140 prior to the gas 3140exiting the atomizing nozzle 310. In other embodiments, the cylindricaltip 3145 extends lower than the annular gas orifice 3150. In still otherembodiments, the cylindrical tip 3145 is slightly recessed within theannular gas orifice 3150, such that the gas 3140 comes into physicalcommunication with the second fluid 3120 prior to the second fluid fullyexiting from the nozzle 310.

In some embodiments, as the gas 3140 passes through the annular gasorifice 3150 and exits the atomizing nozzle 310, it comes in physicalcommunication with the second fluid 3120 and acts to shear the stream ofthe second fluid 3120 and the first fluid 3115 into multiple atomizeddroplets 3155 made of a first fluid 3115 core and a second fluid 3120shell. This configuration of the three fluid nozzle can be best achievedwhen the cross sectional areas of the inner fluid conduit 3165 and thesecond fluid path within the fluid contacting chamber 3125 are chosensuch that the velocities of the first fluid 3115 and the second fluid3120 are approximately equal at the exit of the inner conduit 3163.

In some embodiments, the atomizing nozzle 310 can be configured toproduce very high yield large diameter synthetic membrane vesicles (thatis, vesicles that encapsulate a high amount of a therapeutic agent). Insome embodiments, the large diameter synthetic membrane vesicles can beunilamilar vesicles or multilamilar vesicles or polymer spheres encasinga liquid comprising a therapeutic agent. In some embodiments, the largediameter synthetic membrane vesicles can be multivesicular liposomes. Insome embodiments, the first fluid 3115 can be an aqueous (or similarhydrophilic liquid) phase containing the therapeutic agent and thesecond fluid 3120 can be an organic phase comprising phospholipids orother encasing material e.g. a polymer or PLGA (poly(lactic-co-glycolicacid), biocompatible and in-vivo degradable polymer) or a wax. In someembodiments, the gas 3140 combines with an aqueous phase and an organicphase to afford a droplet of aqueous phase inside a droplet of organicphase, which can, for example, be used to form unilamilar vesicles ormultilamilar vesicles. In some embodiments, the size of the inneraqueous phase droplet can have an average diameter of at least about 0.5μm, 1 μm, 5 μm, 10 μm, 15 μm, 20 μm, or 50 μm, or a diameter within arange defined by any of two of the preceding values. In someembodiments, the organic solvent can be removed by using a normal spraydrying system. In some embodiments, the organic solvent can be removedby using solvent removal chamber such as the one described in FIG. 7.

In some embodiments, the nozzle 310 can comprise an additional outerfluid conduit (not shown), which annularly surrounds the inner conduit3165 to provide a four fluid atomizing nozzle as shown in FIGS. 3E-3L.In some embodiments, the conduit which annularly surrounds the innerconduit 3165 can include an an aqueous phase suitable as a suspendingmedium. In some embodiments, the nozzle can be operated to produce anemulsion where an aqueous drug core can be surrounded by phospholipidsin a volatile solvent that in turn is surrounded by another aqueousphase, which can, for example, be used to form unilamilar vesicles ormultilamilar vesicles. In some embodiments, the large diameter syntheticmembrane vesicles will initially have a hydrophobic outer surface.

In some embodiments, the nozzle 310 can omit the conduit 3123 in orderto provide a two fluid nozzle (not shown). In some embodiments, the twofluids are a liquid and a gas. In some embodiments, the two fluid nozzle(not shown) can be used to spray a first component into the instantsolvent removal chamber such as the one described in FIG. 7. In someembodiments, the organic solvent can be removed by contacting the firstcomponent with an inert gas to in order to create the MVL. In someembodiments excess phospholipids can be used in the first component tocreate the MVL having a coating of phospholipids which initially has ahydrophic surface and can partially shed to afford the MVL with ahydrophilic surface.

FIG. 3E through FIG. 3L

Liposomes with high incorporation percentages (especially important foran expensive active agents) can be made with a four fluid atomizingnozzle 320 as depicted in FIG. 3E through FIG. 3L. In some embodiments,a first fluid 3115 can be a fluid comprising an aqueous active agent,while a second fluid 3170 can be a volatile solvent comprising a lipidsolution, wherein the second fluid 3170 forms a membrane while a thirdfluid 3120 can be a suspending buffer. Without the suspending bufferouter layer, the lipids would for with their hydrophobic regions facingout towards the hydrophobic gas. This four fluid nozzle assembles theouter membrane in a way to prevent aggregation. In such embodiments, theincorporation of the active agent is high because the active agent fluidcan be surrounded by the lipid layer forming solvent 3170. Smallconcentrations of lipids in 3170 can provide at least one bilayer. Insome embodiments, higher concentrations of lipid in the second fluid canprovide liposomes comprising thick large numbers of bilayers. In someembodiments, structurally strong and stable liposomes can be made usingtight packing saturated phospholipids with a high transition temperatureas they are deposited from an evaporating solvent solution and do nothave to be formed in aqueous solutions as per conventional liposomeprocesses.

When the first core fluid 3115 is the first component lacking any lipidsthat could interact with the active agent (e.g. charged lipid like DPPG)and 3170 is volatile solvent (could be same or different that used infirst component) and 3120 is a suspending buffer; MVLs with differentlipids on the outside. These outside lipids (from 3170) can be highlycharged for stability or long chain saturated for mechanical strength.The lipids can be put on the MVL as 1 or 2 bilayers or, if the lipidconcentration in 3170 is high, be put on as a thick mechanically strongmany multiple-bilayers (not previously done) to increase stability ofthe MVL and provide longer slower active agent release. If polymers(e.g. PLGA) were also added to 3120 (w/wo lipids), a polymer skeletoncould be formed surrounding the MVL, further stabilizing it physicallyand chemically. If the polymer layer were strong enough. it would allowthe MVLs to be lyophilized and be stable for very long times at roomtemperature.

FIG. 3E is a schematic partial cross-sectional view of a four fluidatomizing nozzle 320, comprising a first inner fluid conduit (centralneedle) 3165, a second inner conduit 3175, an outer conduit 3123, a gasinput channel 3130, an annular gas chamber (a fourth fluid conduit)3135, a first fluid contacting chamber 3125, a second fluid contactingchamber 3137, and an annular gas orifice 3150. The first fluidcontacting chamber 3137 is conically tapered from the bottom of thefirst inner fluid conduit 3165 and the exit orfice 3147 of the secondinner fluid conduit 3175. The second fluid contacting chamber 3137 isconically tapered from the exit orfice 3147 of the second inner fluidconduit 3175 to a cylindrical tip 3145. In some embodiments, the exitorfice 3147 of the second inner fluid conduit 3175 can be between theexit orfice of the first inner fluid conduit 3163 and the cylindricaltip 3145.

In some embodiments, the second fluid contacting chamber, allows for thefirst and second fluids to contact the third fluid 3120 for a shorterduration than the nozzle described in FIG. 3I. In some embodiments, thefour fluid atomizing nozzle 320 depicted in FIG. 3E can handle flowrates higher than the one fluid contacting chamber nozzle described inFIG. 3I through FIG. 3L due to lower flow instabilities. In someembodiments, the first and second fluid contacting chambers arehydraulic extrusion cones.

The second fluid contacting chamber 3137 is annularly surrounded by theannular gas chamber 3135, which narrows to form an annular gas orifice3150. The exit orfice portion of the cylindrical tip 3145 is annularlyand concentrically surrounded by the annular gas orifice 3150.

In some embodiments, a first fluid 3115 can travel at a volumetric flowrate between 2 mL/minute and 1,000 mL/minute, between 10 mL/minute and500 mL/minute, between 10 mL/minute and 100 mL/minute, and preferablybetween 50 mL/minute and 100 mL/minute through the first inner fluidconduit 3165. In some embodiments, the first fluid exits the exit orfice3163 of the first inner fluid conduit 3165 and enters the first fluidcontacting chamber 3125, whereupon the first fluid 3115 comes inphysical communication with the second fluid 3170. In some embodiments,the second fluid 3170 travels at a volumetric flow rate between 2mL/minute and 1,000 mL/minute, between 10 mL/minute and 500 mL/minute,between 10 mL/minute and 100 mL/minute, and preferably between 50mL/minute and 100 mL/minute, through a second inner fluid conduit 3175.In a typical embodiment, the first fluid 3115 is an emulsion without anylipids that could interact with the active agent (e.g. no charged lipidsuch as DPPG) In another embodiment, the second fluid 3170 is a volatilesolvent comprising a lipid solution (could be the same or different thanthat used in the first component).

In some embodiments, the first fluid 3115 travels through the firstfluid contacting chamber 3125, surrounded by the second fluid 3170. Insome embodiments, the first fluid 3115 and the second fluid 3170continue through the first fluid contacting chamber 3125 into the secondfluid contacting chamber 3137. In some embodiments, the second fluid3170 forms a sheath around the first fluid 3115 core.

In some embodiments, the first and second fluids can travel at avolumetric flow rate between 2 mL/minute and 1,000 mL/minute, between 10mL/minute and 500 mL/minute, between 10 mL/minute and 100 mL/minute, andpreferably between 50 mL/minute and 100 mL/minute through the secondinner conduit 3175. In some embodiments, the first and second fluidsexits the exit orfice 3147 the second inner conduit 3175 and enter thesecond fluid contacting chamber 3137, whereupon the first and secondfluids come in physical communication with a third fluid 3120. In atypical embodiment, the third fluid 3120 is a buffer solution. In someembodiments, the third fluid 3120 travels at a volumetric flow ratebetween 2 mL/minute and 1,000 mL/minute, between 10 mL/minute and 500mL/minute, between 10 mL/minute and 100 mL/minute, and preferablybetween 50 mL/minute and 100 mL/minute, through an outer fluid conduit3123, which annularly surrounds the first inner fluid conduit 3165 andthe second inner conduit 3175 (as seen from the downward perspectiveview F and displayed in FIG. 3F). In some embodiments, the first, secondand third fluids exit the exit orfice 3145 of the second fluidcontacting chamber 3137. In some embodiments, the third fluid 3120 formsa sheath around the first and second fluids.

In some embodiments, the diameter of the first fluid contacting chamber3125 can be conically narrowed as it approaches the exit orfice 3163 ofthe first inner fluid conduit 3165. As the first fluid 3115 travelsthrough the narrowing first fluid contacting chamber 3125 and toward theexit orfice 3147 of the second fluid conduit 3175, the diameter of thefirst fluid 3115 core is correspondingly decreased. Likewise, the secondfluid 3170 can be constricted to create a thinner concentric annularsheath around the first fluid 3115 core. As a result of decreasing thediameter of the first fluid contacting chamber 3125 and passing thefluids through the exit orfice 3147 of the second fluid conduit 3175,the velocities of the first fluid 3115 and the second fluid 3170 areincreased.

In some embodiments, the diameter of the second fluid contacting chamber3125 can be conically narrowed as it joins the cylindrical tip 3145. Asthe first fluid 3115 core and second fluid 3120 travel through thenarrowing second fluid contacting chamber 3125 into the cylindrical tip3145, the diameter of the first fluid 3115 core, the second fluid 3120the third fluid 3170 can be further correspondingly decreased. Likewise,the third fluid 3170 can be constricted to create a thinner concentricannular sheath around the first fluid 3115 core and the second fluid3120 sheath. In some embodiments, the multi vesicular liposomes arelipid coated with lipids on the outside that are different than thelipids on the inside. In some embodiments, the four fluid nozzleillustrated in FIG. 3E and FIG. 3I could be used to manufacture multivesicular liposomes coated with an additional layer of polymer PLGA or ahighly charged phospholipid different from the phospholipid of theinternal membrane. These multi vesicular liposomes coated with an extralayer of polymer or phospholipid or combination thereof constitutefurther embodiments as disclosed herein.

The four fluid atomizing nozzle 320 comprises a gas input channel 3130which supplies a gas 3140. In some embodiments, the volumetric flow rateof the gas can be between 30 L/minute and 1,000 L/minute, between 30L/minute and 500 L/minute, between 20 L/minute and 200 L/minute, andpreferably between 25 L/minute and 100 L/minute and at a pressurebetween 5 psig and 1,000 psig, between 10 psig and 250 psig, between 20psig and 150 psig, and preferably between 50 psig and 120 psig to theannular gas chamber 3135. The above atomizing nozzle flow rates are fora single atomizing nozzle. Prior to reaching the nozzle 320, the gas3140 can be passed through a gas filtration system (not shown)comprising a hydrophobic gas sterilizer (as seen in FIG. 1A and FIG. 1B,component 35). In some embodiments, the outlet of the cylindrical tip3145 and the outlet of the exit orfice 3147 of the first secondcontacting chamber 3137 have the same diameter. In some embodiments, thediameter of the outlet of the cylindrical tip 3145 is larger than thediameter of the outlet of the exit orfice 3147 of the second fluidcontacting chamber 3137. In some embodiments, the diameter of the outletof the cylindrical tip 3145 is smaller than the diameter of the exitorfice 3147 of the second fluid contacting chamber 3137.

FIG. 3G shows an expanded cross-sectional view of the first fluid,second fluid and third fluid in the cylindrical tip 3145.

FIG. 3H shows an expanded cross-sectional view of the first fluid,second fluid and third fluid in a droplet 3255

FIG. 3I

FIG. 3I is a schematic partial cross-sectional view of a four fluidatomizing nozzle 320, comprising a first inner fluid conduit 3165, asecond inner conduit 3175, an outer conduit 3123, a fluid contactingchamber 3125, a gas input channel 3130, an annular gas chamber (a fourthfluid conduit) 3135, and an annular gas orifice 3150. In someembodiments, the fluid contacting chamber 3125 is conically tapered fromthe exit orfice 3173 of an outer fluid conduit 3123 to a cylindrical tip3145.

The fluid contacting chamber 3125 is annularly surrounded by an annulargas chamber 3135, which narrows to form an annular gas orifice 3150. Thebottom portion of the cylindrical tip 3145 is annularly andconcentrically surrounded by the annular gas orifice 3150.

In some embodiments, a first fluid 3115 can travel at a volumetric flowrate between 2 mL/minute and 1,000 mL/minute, between 10 mL/minute and500 mL/minute, between 10 mL/minute and 100 mL/minute, and preferablybetween 50 mL/minute and 100 mL/minute through the first inner fluidconduit 3165. In a typical embodiment, the first fluid 3115 is anemulsion. In some embodiments, the first fluid exits the bottom 3163 ofthe first inner fluid conduit 3165 and enters the fluid contactingchamber 3125, whereupon the first fluid 3115 comes in physicalcommunication with a second fluid 3170. In some embodiments, a secondfluid 3170 exits the bottom 3173 of the second inner conduit 3175 andenters the fluid contacting chamber 3125, whereupon the second fluid3170 comes in physical communication with the first fluid 3115 and athird fluid 3170. In a typical embodiment, the first fluid 3115 in thefirst inner fluid conduit 3165 is annularly surrounded by the secondfluid 3170 in the second inner conduit 3175 which is annularlysurrounded by the third fluid 3120 in the outer conduit 3123 (as seenfrom the downward perspective view J and displayed in FIG. 3J).

In some embodiments, the first fluid 3115 travels through the firstfluid contacting chamber 3125, surrounded by the second fluid 3170 whichis surrounded by the third fluid 3120. In some embodiments, the secondfluid 3170 forms a sheath around the first fluid 3115 and the thirdfluid 3120 forms a sheath around the first and second fluids.

In some embodiments, the diameter of the fluid contacting chamber 3125can be conically narrowed as it joins the cylindrical tip 3145. In someembodiments, as the first fluid 3115 travels through the narrowing firstfluid contacting chamber 3125 and toward the cylindrical tip 3145, thediameter of the first fluid 3115 core is correspondingly decreased. Insome embodiments, the second fluid 3170 can be constricted to create athinner concentric annular sheath around the first fluid 3115 core. Insome embodiments, the third fluid 3120 can be constricted to create athinner concentric annular sheath around the first fluid 3115 core andthe concentric annular second fluid 3170. As a result of decreasing thediameter of the first fluid contacting chamber 3125 and passing thefluids through the cylindrical tip 3145, the velocities of the firstfluid 3115, second fluid 3170 and third fluid 3120 can be increased.

The four fluid atomizing nozzle 320 comprises a gas input channel 3130which supplies a gas 3140. In some embodiments, the volumetric flow rateof the gas can be between 30 L/minute and 1,000 L/minute, between 30L/minute and 500 L/minute, between 20 L/minute and 200 L/minute, andpreferably between 25 L/minute and 100 L/minute and at a pressurebetween 5 psig and 1,000 psig, between 10 psig and 250 psig, between 20psig and 150 psig, and preferably between 40 psig and 120 psig to theannular gas chamber 3135. The above atomizing nozzle flow rates are fora single atomizing nozzle. Prior to reaching the nozzle 320, the gas3140 can be passed through a gas filtration system (not shown)comprising a hydrophobic gas sterilizer (as seen in FIG. 1A and FIG. 1B,component 35).

FIG. 3K shows an expanded cross-sectional view of the first fluid,second fluid and third fluid in the cylindrical tip 3145.

FIG. 3L shows an expanded cross-sectional view of the first fluid,second fluid and third fluid in a droplet 3255.

FIG. 4A

FIG. 4A depicts an atomizing nozzle 410 where the first fluid 4115 canbreak down into large droplets in the fluid contacting chamber 4125prior to reaching the cylindrical tip 4145.

In some embodiments, a first fluid 4115 exits a inner fluid conduit 4165through the exit orfice 4163 of the inner fluid conduit 4165 and entersthe fluid contacting chamber 4125, whereupon the first fluid 4115 comesin physical communication with a second fluid 4120. The second fluid4120 travels through an outer fluid conduit 4123, which annularlysurrounds the inner fluid conduit 4165 (as seen from the downwardperspective view B and displayed in FIG. 4B), to reach the fluidcontacting chamber 4125. The first fluid 4115 forms a plurality of firstfluid droplets 4157 traveling through the fluid contacting chamber 4125,the first fluid droplets 4157 now being in physical communication with,and surrounded by, the second fluid 4120. In some embodiments, the firstand second fluid are immiscible. In a typical embodiment, the firstfluid 4115 is an emulsion and the second fluid 4120 is a buffersolution.

In some embodiments, the diameter of the fluid contacting chamber 4125is conically narrowed as it joins the cylindrical tip 4145. As the firstfluid 4115 travels through the narrowing fluid contacting chamber 4125and into the cylindrical tip 4145, the first fluid droplets 4157 aresqueezed and their diameter is correspondingly decreased along the axisof travel. Likewise, the second fluid 4120 is constricted to create athinner shell around the first fluid droplets 4157. As a result ofdecreasing the diameter of the fluid contacting chamber 4125 and passingthe solutions through the cylindrical tip 4145, the velocities of thefirst fluid droplets 4157 and second fluid 4120 are increased. In atypical embodiment, the first fluid 4115 is an emulsion and the secondfluid 4120 is a buffer solution.

FIG. 4C shows an expanded cross-sectional view of the first fluiddroplets 4157 and second fluid 4120 in the cylindrical tip 4145.

FIG. 4D shows an expanded cross-sectional view of the first fluid andsecond fluid in a droplet 4155. In some embodiments, the ratio of thediameters of the first fluid to the second fluid in a droplet 4155 canbe the same as the ratio of the diameters of the first fluid in theinner fluid conduit 4165 to the second fluid in the outer fluid conduit4123. In some embodiments, the ratio of the diameters of the first fluidto the second fluid in a droplet 4155 can be the larger than the ratioof the diameters of the first fluid in the inner fluid conduit 4165 tothe second fluid in the outer fluid conduit 4123. In some embodiments,the ratio of the diameters of the first fluid to the second fluid in adroplet 4155 can be the smaller than the ratio of the diameters of thefirst fluid in the inner fluid conduit 4165 to the second fluid in theouter fluid conduit 4123.

The nozzle 410 comprises a gas input channel 4130 which supplies a gas4140 to an annular gas chamber (a third fluid conduit) 4135. Prior toreaching the nozzle 410, the gas 4140 can be passed through a gasfiltration system (not shown) comprising a hydrophobic gas sterilizer(FIG. 1A and FIG. 1B, component 35). The wall of the fluid contactingchamber 4125 provides a barrier between the annular gas chamber 4135 andthe fluid contacting chamber 4125 such that there is no physical contactbetween the fluids in the fluid contacting chamber 4125 and the gas 4140in the annular gas chamber 4135. In some embodiments, the outlet of thecylindrical tip 4145 and the outlet of the annular gas orifice 4150 canbe flush with each other such that the second fluid 4120 does not comeinto contact with the gas 4140 prior to the gas 4140 exiting theatomizing nozzle 410. In other embodiments, the cylindrical tip 4145 mayextend below the end of the annular gas orifice 4150. In still otherembodiments, the cylindrical tip 4145 may be slightly recessed withinthe annular gas orifice 4150, such that the gas 4140 comes into physicalcommunication with the second fluid 4120 prior to the second fluid fullyexiting from the nozzle 410. In a typical embodiment, the first fluid4115 is an emulsion and the second fluid 4120 is a buffer solution.

In some embodiments, as the gas 4140 passes through the annular gasorifice 4150 and exits the atomizing nozzle 410, it comes in physicalcommunication with the second fluid 4120 and acts to shear the stream ofthe second fluid 4120 and the first fluid 4115 droplets into atomizeddroplets 4155 made of a first fluid 4115 core and a second fluid 4120shell. In this embodiment the first fluid core stream breaks up due tothe mismatch of velocities between the first fluid 4115 and the secondfluid 4120 at the exit orfice 4163 of the inner fluid conduit 4165. In atypical embodiment, the first fluid 4115 is an emulsion and the secondfluid 4120 is a buffer solution.

FIG. 5

FIG. 5 is a more detailed view of one embodiment of an atomizing nozzle505. The nozzle depicted in FIG. 5 is derived from Part No.1/8JJN-SS+SUJ1A-SS manufactured by Spraying Systems Co. of Wheaton,Ill., as modified in FIG. 5 and described herein. The atomizing nozzlelower section 510 of FIG. 5 provides three fluid input channels for theintroduction of three fluids into the nozzle 505. A first fluid inputchannel is the central longitudinal channel 5114, which extendssubstantially downwardly through the atomizing nozzle lower section 510and extends into a fluid contacting chamber 5125. In some embodiments, asecond fluid input channel is a buffer input channel 5128 and a thirdfluid input channel is a gas input channel 5130.

The central longitudinal channel 5114 is a channel which may be ofcircular cross-section. The central longitudinal channel 5114 may beginas an aperture 5111 in a line coupler 5113. The line coupler 5113 is ameans for attaching a supply line to the nozzle 505 for the introductionof the first fluid 5115 into the nozzle lower section 510. A polymericsealing washer 5112 is retained in contact with the line coupler 5113and interposed between a coupler nut 5116 of the line coupler 5113 and ahead (not shown) of a inner fluid conduit 5165.

The inner fluid conduit 5165 can provide a continuation of the centrallongitudinal channel 5114, with a narrowed central bore 5164 extendingthrough a removable housing member 5170 into the horizontal channelcasing 5175, and into the liquid fluid cap 5180, ending in the fluidcontacting chamber 5125 of the liquid fluid cap 5180. In someembodiments, the housing member 5170 can be threaded. The housing member5170 can be fitted on the top with a housing cap 5172 and housing gasketmember 5171 to provide secure fastening and fluid seal. The housingmember 5170 extends downwardly into a top portion of the horizontalchannel casing 5175, wherein it can be tightened to form a secureconnection with the horizontal channel casing 5175, for example usinginternal screw threading.

In addition to the central longitudinal channel 5114, the horizontalchannel casing 5175 can be comprised of the gas input channel 5130 andthe buffer input channel 5128. The gas input channel 5130 can have anexternal aperture 5131 to which a gas supply line (not shown) can be tobe attached. The gas input channel 5130 can have an internal aperture5132 which leads to an annular gas chamber (third fluid conduit) 5135,the annular gas chamber 5135 situated to annularly surround the liquidfluid cap 5180. The liquid fluid cap 5180 can be detachably and securelyfitted into the horizontal channel casing 5175 such that fluid contentsof the liquid fluid cap 5180 and the fluid contacting chamber 5125cannot physically communicate with the fluid contents of the annular gaschamber 5135 inside the nozzle. The buffer input channel 5128 can havean external aperture 5127 to which a fluid supply line (not shown) canbe attached. The buffer input channel 5128 can have an internal aperture5126 which leads to the top opening of the liquid fluid cap 5180.

In some embodiments, the liquid fluid cap 5180 can be cylindricallyshaped in a top portion and conically tapered in a exit orfice portion.In some embodiments, the liquid fluid cap 5180 can be cylindricallytapered in both a top portion and a bottom portion. In some embodiments,the fluid contacting chamber 5125 and the outer fluid conduit 5123 formthe interior wall of the liquid fluid cap 5180. The fluid contactingchamber 5125 is conically tapered from the exit orfice of an annularouter fluid conduit 5123 to a cylindrical tip 5145. The exit orficeportion of the cylindrical tip 5145 can be annularly and concentricallysurrounded by an annular gas orifice 5150. The annular gas orifice 5150can be created by placement of the cylindrical tip 5145 in the center ofa cylindrical opening in a gas cap 5160. The liquid fluid cap 5180 canhave a gas restrictor 5182 attached to the exterior of the liquid fluidcap 5180 with several circular holes in the gas restrictor 5182, whichannularly surrounding the liquid fluid cap 5180. The presence of the gasrestrictor 5182 creates a lower annular gas chamber 5136 between the gasrestrictor 5182 and the gas cap 5160. The gas restrictor 5182 canminimize the turbulence possibly created as a gas travels through theannular gas chamber 5135 to the annular gas orifice 5150. The nozzlelower section 510 can be fitted to an orifice housing 5185 which acts tokeep the gas cap 5160 and liquid fluid cap 5180 securely fitted to thehorizontal channel casing 5175. In some embodiments, the orifice housing5185 can be threaded.

In some embodiments, the first fluid 5115 can enter the longitudinalchannel 5114 of the nozzle lower section 510 from a supply line (notshown) attached to the line coupler 5113. The first fluid travelsthrough the longitudinal channel 5114 until it reaches the fluidcontacting chamber 5125 whereupon it comes in physical contact with thesecond fluid 5120 in the fluid contacting chamber 5125. In a typicalembodiment, the first fluid 5115 is an emulsion and the second fluid5120 is a buffer solution.

In some embodiments, the second fluid 5120 enters the nozzle lowersection 510 through an input aperture 5127 and travels through the inputchannel 5128. In some embodiments, the second fluid 5120 then passesthrough the internal aperture 5126 leading to the top of the liquidfluid cap 5180 and travels through the annular outer fluid conduit 5123,and travels around and surrounds the inner fluid conduit 5165 at a lowersection to reach the fluid contacting chamber 5125. In some embodiments,upon exiting the exit orfice 5163 of the inner fluid conduit 5165 or thecentral longitudinal channel 5114 and entering the fluid contactingchamber 5125, the first fluid 5115 can form a cylindrical core travelingthrough the fluid contacting chamber 5125. In some embodiments, thefirst fluid 5115 cylindrical core can be surrounded annularly by thesecond fluid 5120. In some embodiments, the immiscibility between thesecond fluid 5120 and the first fluid 5115, and because of the highvelocity at which the second fluid 5120 and first fluid 5115 aretraveling, causes the second fluid 5120 to form a sheath around thefirst fluid 5115 core. The first fluid 5115 and second fluid 5120 cantravel through the fluid contacting chamber 5125 into the cylindricaltip 5145 of the outer conduit 5123. In a typical embodiment, the firstfluid 5115 is an emulsion and the second fluid 5120 is a buffersolution.

The cross-sectional diameter of the fluid contacting chamber 5125decreases as it conically narrows to join the cylindrical tip 5145. Asthe first fluid 5115 travels through the narrowing fluid contactingchamber 5125 and into the cylindrical tip 5145 the diameter of the firstfluid 5115 core is decreased. Likewise, the second fluid 5120 can beconstricted to create a thinner concentric annular sheath around thefirst component 5115 core. In some embodiments, as the solutions passthrough the narrowing cylindrical tip, the velocities of the first fluid5115 and the second fluid 5120 can be increased. In some embodiments,the threads on the inner fluid conduit 5165 and the housing member 5170allow the outlet tip of the inner fluid conduit 5165 to be adjustedvertically within the conical portion of the fluid contacting chamber5125 within the liquid fluid cap 5180. In some embodiments, the housingmember 5170 can be adjusted so that the velocities of the second fluid5120 and the first fluid 5115 can be made approximately equal at thepoint where the first fluid 5115 exits the inner fluid conduit 5165.This allows optimal operation as shown in FIG. 3A. In some embodiments,the housing member 5170 can be adjusted so that the velocities of thesecond fluid 5120 and the first fluid 5115 can be made uequal at thepoint where the first fluid 5115 exits the inner fluid conduit 5165.This allows optimal operation as shown in FIG. 4A. In a typicalembodiment, the first fluid 5115 is an emulsion and the second fluid5120 is a buffer solution.

In some embodiments, a gas 5140 can be first passed through a gasfiltration system (not shown) comprising a hydrophobic gas sterilizer(not shown). In some embodiments, the gas 5140 can enter the gas inputchannel 5130 through the external gas input aperture 5131 and thenpasses through the internal gas chamber aperture 5132 to reach theannular gas chamber 5135. In some embodiments, the liquid fluid cap 5180can be detachably and securely fitted into the horizontal channel casing5175 such that the gas 5140 cannot physically communicate with the firstfluid 5115 or the second fluid 5120 inside the nozzle lower section 510.The gas 5140 travels through the holes in the gas restrictor 5182 toreach the lower annular gas chamber 5136.

As the gas 5140 passes through the annular gas orifice 5150 and exitsthe atomizing nozzle lower section 510, it comes in physicalcommunication with the second fluid 5120 and acts to shear the stream ofthe second fluid 5120 and first fluid 5115 into atomized droplets 5155made of a first fluid 5115 core and a second fluid 5120 shell. In atypical embodiment, the first fluid 5115 is an emulsion and the secondfluid 5120 is a buffer solution. In a typical embodiment, the firstfluid 5115 core is an emulsion and the second fluid 5120 shell is abuffer solution.

Some embodiments provide an atomizing nozzle apparatus, comprising threechannels, each having at least one entrance orifice and one exitorifice, the channels comprise of an inner fluid conduit and an outerfluid conduit, wherein the exit orifice for the inner fluid conduit isof a diameter smaller than, and is located centrally in, the outer fluidconduit and is directed towards the outer fluid conduit exit orifice, agas channel, wherein a pressurized gas exiting the gas channel exitorifice impinges a liquid exiting the exit orifice of the outer fluidconduit. Additional embodiments include the processes for using such adevice and the MVL products made by the same. In some embodiments, thegas channel exit orifice can annularly surround the exit orifice of theouter fluid conduit. In some embodiments, the gas channel exit orificeand the outer fluid conduit exit orifice can be flush. In someembodiments, the outer fluid conduit exit orifice can be recessed withinthe gas channel exit orifice. In some embodiments, the outer fluidconduit exit orifice can extend beyond the gas channel exit orifice. Insome embodiments, the exit orifices of all three channels can becoaxial. In some embodiments, the exit orifice of the inner fluidconduit can be more than two outer fluid conduit exit orifice diametersaway from the outer fluid conduit exit orifice.

In some embodiments, the multi vesicular liposome product produced bythe atomizing nozzle in FIGS. 3-6 can be directly collected andpurified. Alternatively, the MULTI VESICULAR LIPOSOME product can beprocessed by an evaporation tower or the evaporation apparatus of FIG. 7(or as part of FIGS. 1A-1C) before being processed.

FIG. 6

FIG. 6 is an exploded schematic 605 of the individual components of thedevice of FIG. 5.

In some embodiments, component 5113 is a line coupler; component 5165 isan inner fluid conduit; component 5172 is a housing cap; component 5170is a housing member; component 5171 is a housing gasket member;component 5180 is a liquid fluid cap; component 5145 is a cylindricaltip; component 5160 is a gas cap; component 5182 is a gas restrictor;and component 5185 is an orifice housing.

In some embodiments, location 5130 can be a gas input channel; location5132 can be an internal gas chamber aperture; location 5128 can be afluid channel; location 5126 can be an internal fluid cap aperture; andlocation 5150 can be an annular gas orifice.

FIG. 7

An embodiment of the present application is a system for manufacturingformulations including an evaporation apparatus, or evaporationsub-system, the process of using the system, and the large diametersynthetic membrane vesicles products made by the process. An example ofan evaporation apparatus is the solvent removal vessel presented in FIG.7 and described herein. FIG. 7 shows a schematic of a solvent removalvessel 710. In some embodiments, atomized droplets 7155 can be sprayedinto the solvent removal vessel 710 in order to remove solvent fromatomized droplets 7155. In some embodiments, the solvent removal vessel710 can include a three-fluid atomizing nozzle 7510, described above,attached to and extending through a lid 7220 of the solvent removalvessel 710. In some embodiments, the cylindrical tip and annular gasorifice of the atomizing nozzle 7510 can be configured to besubstantially flush with the inside edge of the lid 7220. In someembodiments, the cylindrical tip and annular gas orifice of theatomizing nozzle 7510 can be configured to extend beyond the inside edgeof the lid 7220.

Alternatively, in some embodiments, the solvent removal vessel 710 caninclude a three-fluid atomizing nozzle 7510, described above, attachedto and extending through a wall 7350 of the solvent removal vessel 710.In some embodiments, the cylindrical tip and annular gas orifice of theatomizing nozzle 7510 can be configured to be substantially flush withthe inside edge of the wall 7350. In some embodiments, the cylindricaltip and annular gas orifice of the atomizing nozzle 7510 can beconfigured to extend beyond the inside edge of the wall 7350.

In some embodiments, the vessel 710 also includes a gas outlet tube7340, which can pass through the center of the lid 7220, and extend intoa solvent removal chamber 7230. In some embodiments, the gas outlet tube7340 can extend down from the lid from about 15 to about 19 inches andcan be about 1 inch in diameter. In some embodiments, the gas outlettube 7340 can extend down from the lid from about 20 cm to about 60 cmand can be from about 1 cm to about 4 cm in diameter. In someembodiments, a conical fitting 7300 can be attached to the end of thegas outlet tube 7340. In some embodiments, the conical fitting 7300 canbe used to narrow the diameter of the gas outlet tube 7340 at a gasoutlet 7310. In some embodiments, the gas outlet 7310 can beapproximately 0.5 inches in diameter. In some embodiments, the gasoutlet 7310 can be from about 0.5 cm to about 3 cm in diameter. In someembodiments, a vortex stabilizer disk or ring 7360 can be attached tothe conical fitting 7300.

In some embodiments, a gas inlet pipe 7290 can be connected to the wall7350 of the solvent removal vessel 710 can be creating a gas inlet 7280.In some embodiments, the solvent removal vessel 710 has a bottom 7250,which can include a product exit orifice 7260. In some embodiments, theexit orifice 7260 can be in the center of the domed bottom 7250. In someembodiments, a product outlet pipe 7270 can be attached to the exitorifice 7260. In some embodiments, the solvent removal vessel 710,includes a temperature control jacket (not shown). In some embodiments,temperature controlling fluid can be circulated through a temperaturecontrol jacket (not shown) of the solvent removal vessel 710, whereinthe temperature control jacket can surround the wall 7350 and the bottom7250 of the solvent removal vessel 710 for temperature regulation in thesolvent removal chamber 7230. In some embodiments, the bottom 7250 ofthe solvent removal vessel 710 could be of a shape such as a dome, acone or an angled flat bottom.

In some embodiments, a carrier gas 7370 can be first heated andhumidified and then passed through a gas filtration system (not shown)comprising a hydrophobic gas sterilizer (not shown). In someembodiments, the carrier gas 7370 can be supplied to the solvent removalvessel 710 through the gas inlet pipe 7290. In some embodiments, thecarrier gas 7370 can pass through the gas inlet 7280 and enter a gasrotation jet 7285. In some embodiments, the gas rotation jet 7285directs the flowing carrier gas 7370 inside the solvent removal chamber7230 such that the carrier gas 7370 exits the gas rotation jet 7285 (andenters the solvent removal chamber 7230) horizontally and in a directiontangential to the solvent removal vessel wall 5350. In some embodiments,substantially all the carrier gas 7370 entering the solvent removalchamber 7230 through the gas rotation jet 7285 can exit the solventremoval chamber 7230 through the gas outlet 7310 and gas outlet pipe7340.

In some embodiments, the carrier gas 7370 supplied to solvent removalchamber 7230 can be injected tangential to the wall 7350, causing thecarrier gas 7370 to first travel slowly clockwise (as viewed from above)around the solvent removal vessel 710 near the wall 7350, and forming aslow gas rotation 7240. In some embodiments, the solvent removal chamber7230 can be pressured to approximately 1 psig, creating a pressuredifferential between the solvent removal chamber 7230 and the gas outletpipe 7340. In some embodiments, the carrier gas 7370 traveling in thesolvent removal chamber 7230 can be pulled inwards, as it circulates,towards the gas outlet 7310. In some embodiments, the change in angularmomentum on the carrier gas 7370 causes the carrier gas 7370 toaccelerate as it moves closer to the gas outlet 7310. In someembodiments, the acceleration of the carrier gas 7370 near the gasoutlet 7310 can be sufficiently strong to create an intense gas vortex7245 underneath the vortex stabilizer 7360 and gas outlet 7310. In someembodiments, the carrier gas 7370 can be pushed out of the solventremoval chamber 7230 through the gas outlet 7310 into the gas outletpipe 7340 for disposal after spinning through the gas vortex 7245.

In some embodiments, the three-fluid atomizing nozzle 7510 can besupplied with a first fluid 7115, a second fluid 7120, and a third fluid7140, as described above. In a typical embodiment, the first fluid canbe a first component, the second fluid can be a buffer solution, and thethird fluid can be a gas. In some embodiments, the resultant atomizeddroplets 7155, comprised of a first fluid core and a second fluid shell,can be sprayed into the solvent removal chamber 7230. In someembodiments, the atomized droplets 7155 come into contact with thecarrier gas 7370 being circulated in the slow gas rotation 7240 as theatomized droplets 7155 can travel down through the solvent removalchamber 7230. In some embodiments, the atomized droplets 7155 can bepicked up by, and incorporated in, the slow gas rotation 7240 and beginto circulate through the solvent removal chamber 7230, and begin tosettle toward the domed bottom 7250.

In some embodiments, the solvent can be substantially evaporated fromthe core of the atomized droplets 7155 as the atomized droplets 7155circulate along with the carrier gas 7370 in the slow gas rotation 7240.In some embodiments, the evaporated solvent can be removed from thesolvent removal chamber 7230 along with the circulating carrier gas 7370through the gas outlet 7310 and into the gas outlet pipe 7340 fordisposal. In some embodiments, the gas outlet pipe 7340 can be equippedwith a sterile barrier filtration system (not shown) outside of solventremoval vessel 710. The filtration system in one embodiment can becomprised of filter (e.g. a course filter or conventional cycloneseparator) and a HEPA filter or other sterilizing gas filter.

In some embodiments, a portion of the atomized droplets 7155, travelingin the slow gas rotation 7240, can reach the intense gas vortex 7245,where they can be kicked outwards again by the intense centrifugalforces within the intense gas vortex 7245, and thus are not removedthrough the gas outlet 7310. For example, some of the atomized droplets7155 kicked outwards by the gas vortex 7245 can travel again through theslow gas rotation 7240 and some of the atomized droplets begin to falltowards the domed bottom 7250. In one embodiment, very few of theatomized droplets 7155 escape with the carrier gas 7370 through the gasoutlet 7310. In some embodiments, removal of substantially all thesolvent from the atomized droplets 7155 can afford large diametersynthetic membrane vesicles droplets 7380 still coated in a fluid shell.In some embodiments, droplets 7380 permanently fall out of the gasvortex primarily to the domed bottom 7250 of the solvent removal vessel710. In some embodiments, the plurality of large diameter syntheticmembrane vesicles droplets 7380 form a suspension 7390 of large diametersynthetic membrane vesicles particles in a solution at the domed bottom7250. In a typical embodiment, the large diameter synthetic membranevesicles are multivesicular liposomes.

The solvent removal vessel 710 can optionally be equipped with atwo-fluid rinse nozzle 7400 in the lid 7220 of the solvent removalvessel 710. In some embodiments, the two-fluid rinse nozzle 7400 can bepositioned in the lid 7220 at 90 degrees clockwise from the atomizingnozzle 7510. In some embodiments, the two-fluid rinse nozzle 7400 can bepositioned in the lid 7220 at 180 degrees clockwise from the atomizingnozzle 7510. The rinse nozzle 7400 can be fed a wall rinse solution 7410and a filtered gas 7420 in order to spray atomized wall rinse solutiondroplets 7415 into the solvent removal vessel 710 for the purpose ofrinsing any wayward atomized emulsion droplets from the wall 7350 andrinsing the large diameter synthetic membrane vesicles suspension 7390to the product exit orifice 7260. In a typical embodiment, the largediameter synthetic membrane vesicles are multivesicular liposomes.

In some embodiments, solvent removal vessel 710 can be optionallyequipped with a lid-protecting gas inlet 7430 in the lid 7220. In someembodiments, solvent removal vessel 710 can be optionally equipped witha lid-protecting gas inlet 7430 in the lid 7220 on a side opposite theatomizing nozzle 7510. In some embodiments, humidified, sterilized, andfiltered gas 7440 can be streamed into the solvent removal chamber 7230in a direction tangential to the solvent removal vessel wall 7350through the lid-protecting gas inlet 7430, near the top of the solventremoval chamber 7230. In some embodiments, gas 7440 can travelcircularly, as a lid protection jet, around the top of solvent removalchamber 7230 and, in essence, acts a gas cushion above the gas vortex7245 and slow gas rotation 7240 to prevent droplet buildup on the lid7220. In some embodiments, the gas streamed through the lid-protectinggas inlet can be, for example, nitrogen gas (or a nitrogen gas/aqueousvapor mixture) or air scrubbed of CO₂. In some embodiments, the carriergas can be nitrogen gas (or a nitrogen gas/aqueous vapor mixture) or airscrubbed of CO₂.

In some embodiments, the vortex stabilizer 7360 can be a metal disk orring extending radially outward from the conical fitting 7300 and can befitted flush with the gas outlet 7310 at the end of the conical fitting7300. In some embodiments, the diameter of the vortex stabilizer 7360 istwice to six times the diameter of the gas outlet 7310. In someembodiments, the vortex stabilizer 7360 acts to ensure helical stabilityand integrity of the intense gas vortex 7245 by protecting the tip ofthe intense gas vortex 7245 from the turbulence caused by the sprayemanating from the atomizing nozzle 7510. In some embodiments, thestability of the gas vortex 7245 can be maintained by placement of theatomizing nozzle 7510 at a distance between ¼ lid radius from the wall7350 and ¼ lid radius from the gas outlet pipe 7340. In someembodiments, the atomizing nozzle 7510 can be positioned at a distanceof 7/11 the radius of the lid 7220 from the gas outlet pipe 7340.Strategic placement of the atomizing nozzle 7510 minimizes the impact onthe intense gas vortex 7245 of spraying the atomized droplets 7155 intothe solvent removal chamber 7230 and also minimizes the number of theatomized droplets 7155 that impinge on the wall 7350. In someembodiments, the rinse nozzle 7400 placement is not critical as long asa substantial quantity of rinse solution droplets 7415 enter the slowgas rotation 7240 within the solvent removal vessel 7240 and deposit onthe vessel walls 7350 and bottom 7250. In some embodiments, the rinsenozzle 7400 can also be of a one fluid (liquid) design fed bypressurized rinse solution. In some embodiments, the nozzle can be 180degrees from the three fluid nozzle to suppress off access gas rotation.

In some embodiments, the suspension 7390 collected at the domed bottom7250 of the solvent removal vessel 710 can be drained and optionallypumped by a pump (not shown) from the domed bottom 7250 through theproduct exit orifice 7260 into the product outlet pipe 7270 tooptionally be further processed. In some embodiments, the suspension7390 can be further processed by a buffer exchange through a series ofdiafilters.

In some embodiments, the solvent removal vessel of FIG. 7 can be acomponent of FIGS. 1A and B (component 50). In some embodiments, theatomizing nozzle 7510 can receive fluid from the line 2180 of FIG. 2. Insome embodiments, the large diameter synthetic membrane vesiclessuspension 7390 can exit the solvent removal vessel of FIG. 7 throughthe product exit orifice 7260 and can enter the systems of FIG. 1C(through line 120), FIG. 8 (through line 8120), FIG. 10 (through line0120) and/or FIG. 11 (through line 1120). Alternatively, the largediameter synthetic membrane vesicles suspension 7390 can be collecteddirectly from the product exit orifice 7260.

In some embodiments, the apparatus is configured where the gas inlet canbe situated in the chamber to cause the gas to rotate within the chamberaround an axis of the chamber and a small diameter gas exit orificesituated on that same axis and adapted to form a small diameter, inrelation to the tank diameter, intense rapidly rotating gas vortexcapable of substantially preventing atomized droplets from exitingthrough the gas exit.

In some embodiments, the apparatus is configured where the gas inlet,tank dimensions and gas exit diameter and position are together adaptedto retain the atomized droplets in the circulating gas stream ratherthat force them to a wall. In some embodiments, the atomized dropletscan build up in the circulating gas stream until the rate of settling tothe liquid exit equals the rate of generation by the atomizing nozzle.

Some embodiments provide an evaporation apparatus, comprising a sealedsolvent removal vessel, comprised of a lid, a bottom, and a circularwall, at least one atomizing nozzle which is not located on the centralaxis of the circular wall, a carrier gas entrance orifice tangential tothe circular wall, a carrier gas exit orifice located on the centralaxis of the circular wall and directed along the axis with a diameter ofless than ⅕ of the circular wall diameter, and a product exit orifice inthe bottom of the vessel. Additional embodiments include the processesfor using such a device and the MVL products made by the same. In someembodiments, at least part of the vessel can be jacketed. In someembodiments, the atomizing nozzle can be mounted to and extendingthrough the lid of the solvent removal vessel. In some embodiments, theapparatus further comprises a rinse nozzle mounted to and extendingthrough the lid of the solvent removal vessel. In some embodiments, theatomizing nozzle can be used to cause the gas within the vessel torotate. In some embodiments, the atomizing nozzle can be angled at least5 degrees measured off the central axis of the wall and in a planeparallel to the wall nearest to it. In some embodiments, the gasentrance orifice can be combined with the atomizing nozzle. In someembodiments, the carrier gas exit orifice can comprise a tube extendingapproximately ⅔ of the way into the solvent removal vessel. In someembodiments, the tube can be fitted with a narrowing cone and anoptional annular ring. In some embodiments, the gas exit orificediameter can be less than a 1/10 diameter of the diameter of the insideof the tank. In some embodiments, the atomizing nozzle can be a nozzleas disclosed herein. In some embodiments, the ratio of the insidediameter of the solvent removal vessel to the diameter of the carriergas exit orifice can be between approximately 5:1 and 100:1. In someembodiments, the ratio of the inside diameter of the solvent removalvessel to the diameter of the carrier gas exit orifice can be betweenapproximately 20:1 and 60:1. In some embodiments, the solvent removalvessel is comprised of two, three, or four atomizing nozzles which areused to spray the atomized droplets into the solvent removal vessel.

FIG. 8

In some embodiments, the large diameter synthetic membrane vesiclessuspension resulting from the spray evaporation process may optionallyundergo a filtration and/or concentration process in a continuous-flowparticle-concentration system to concentrate the large diametersynthetic membrane vesicles and remove the buffer solution. Oneembodiment is a continuous-flow particle-concentration unit, as well asthe process to concentrate and/or filter the large diameter syntheticmembrane vesicles particles so produced. In a typical embodiment, thelarge diameter synthetic membrane vesicles are multivesicular liposomes.Other embodiments of particle-concentration systems include the use ofone or more hydro-cyclones or one or more centrifuges. An example of ahydro-cyclone is the Minicyclone or Minicyclone Array available fromChemIndustrial Systems, Inc. of Cedarburg, Wis. An example of a diskcentrifuge is Model No. Pathfinder SC1-06-177 manufactured by GEAWestfalia Separator of Oelde, Germany.

In this document the term concentration unit, concentration apparatus,concentration system, particle-concentration system,particle-concentrating device, and particle concentrator are meant toencompass units and processes that remove some or all of the particlesuspending medium of a particle suspension and therefore concentrate theparticles. Furthermore, the definition of these terms encompasses theexchange of the suspending medium with a new suspending medium,performed in one step or incrementally. These two processes are closelyrelated as exchanging the suspending medium can be accomplished byconcentrating the suspension and adding new suspending medium. Theseterms relate to concentrating the particle suspension and exchanging thesuspending medium done separately or simultaneously.

An embodiment of the present application is a system for manufacturingformulations including a continuous-flow diafiltration system, theprocess of using the system, and the large diameter synthetic membranevesicles made by the process. An example of a continuous-flowdiafiltration system is presented in FIG. 8 and described herein. FIG. 8is a schematic of one example of a continuous-flow diafiltration system810. In some embodiments, the permeate discarded in the steps describedherein contains a buffer solution in which large diameter syntheticmembrane vesicles can be suspended as the large diameter syntheticmembrane vesicles suspension exits the solvent removal vessel. In atypical embodiment, the large diameter synthetic membrane vesicles aremultivesicular liposomes.

In some embodiments, the large diameter synthetic membrane vesiclessuspension can be pumped from the solvent removal vessel, such as theexample vessel depicted in FIG. 7 and described above, to thediafiltration system 810 through a particle suspension inlet line 8120(also seen in FIG. 1A, component 120 and in FIG. 7, component 7270),reaching a first retentate vessel 8100. In some embodiments, for each 1L of large diameter synthetic membrane vesicles suspension fed to thefirst retentate vessel 8100, a 2 L solution can be fed into the firstretentate vessel 8100 through a line 8130, the solution first passingthrough a manual valve 8212 and a sterilizing hydrophilic filter 8170.In some embodiments, the large diameter synthetic membrane vesicles aremultivesicular liposomes and the solution is a saline solution.

In some embodiments, a portion of the large diameter synthetic membranevesicles suspension in the first retentate vessel 8100 can be pumped bypump 8110 through a cross-flow (tangential-flow) filtration module 8150.For example, this can be a hollow fiber type module. In one embodiment,the hollow fiber filter used is Model No. CFP-2-E-8A (0.2 micron)manufactured by Amersham Biosciences of Westborough, Mass. In someembodiments, the pore size of the cross-flow (tangential-flow)filtration modules 8150, 8152 and 8154, can be chosen to retain thelarge diameter synthetic membrane vesicles while allowing the suspendingmedium to pass through the filter membranes as permeate. The pumps usedin the diafiltration system can be of various types, such as peristalticor rotary lobe positive displacement pumps. In some embodiments,cross-flow recirculation pumps 8110, 8112 and 8114 operate with at leasttwice the permeate flow rate of their associated cross-flow filtermodule and preferably 3 times, 5 times or 10 times the permeate flowrates. In some embodiments, the permeate can be drawn off through apermeate line 8160 (passing through a sterilizing hydrophilic filter8190 and a manual valve 8202), wherein for each 1 L of large diametersynthetic membrane vesicles suspension added to the first retentatevessel 8100, 2.25 L of permeate can be removed and discarded. In someembodiments, the retentate from the filtration module 8150 can becirculated back into the first retentate vessel 8100 via a retentateline 8140. In some embodiments, for each 1 L of large diameter syntheticmembrane vesicles suspension added to the first retentate vessel 8100, a0.75 L flow of the concentrated large diameter synthetic membranevesicles suspension can be removed from the first retentate vessel 8100through a feed line 8122 and a metering pump 8123 to be further filteredin a second retentate vessel 8200. In some embodiments, the largediameter synthetic membrane vesicles suspension exiting the firstretentate vessel 8100 can be concentrated by a factor of 1.33 (largediameter synthetic membrane vesicles concentration is increased by 33%).In a typical embodiment, the large diameter synthetic membrane vesiclesare multivesicular liposomes.

In some embodiments, similar filtration can occur in the secondretentate vessel 8200 as described above for the first retentate vessel8100. In some embodiments, the large diameter synthetic membranevesicles suspension can enter the second retentate vessel 8200 at a rateof 0.75 L per 1 L added to the first retentate vessel 8100. In someembodiments, a solution can be fed into the second retentate vessel 8200through a line 8132, the solution first passing through a manual valve8204 and a sterilizing hydrophilic filter 8172, at a rate of 2 L per0.75 L concentrated large diameter synthetic membrane vesiclessuspension added to the second retentate vessel 8200. In someembodiments, a portion of the large diameter synthetic membrane vesiclessuspension in the second retentate vessel 8200 can be pumped by pump8112 through a cross-flow (tangential-flow) filtration module 8152. Insome embodiments, the permeate can be drawn off through a permeate line8162 (passing through a sterilizing hydrophilic filter 8192 and a manualvalve 8206), wherein for each 0.75 L of large diameter syntheticmembrane vesicles suspension added to the second retentate vessel 8200,2.25 L of permeate can be removed and discarded. In some embodiments,the retentate from the filtration module 8152 can be circulated backinto the second retentate vessel 8200 via a retentate line 8142. In someembodiments, for each 0.75 L of large diameter synthetic membranevesicles suspension added to the second retentate vessel 8200, a 0.50 Lflow of the concentrated large diameter synthetic membrane vesiclessuspension can be removed from the second retentate vessel 8200 througha feed line 8124 and a metering pump 8125 to be further filtered in afinal retentate vessel 8300. In some embodiments, the large diametersynthetic membrane vesicles suspension exiting the second retentatevessel 8200 can now be concentrated 200%, whereas the buffer solutionconcentration in the large diameter synthetic membrane vesiclessuspension can be roughly 9.1% with respect to the large diametersynthetic membrane vesicles suspension in the inlet line 8120.

In some embodiments, similar filtration can occur in the final retentatevessel 8300 as described above for the first retentate vessel 8100 andsecond retentate vessel 8200. In some embodiments, the concentratedlarge diameter synthetic membrane vesicles suspension traveling throughthe feed line 8124 can enter the final product vessel 8300 where it isfurther filtered and concentrated to contain all the large diametersynthetic membrane vesicles entering through the inlet line 8120, in thefinal product vessel 8300. In some embodiments, a solution can be fedinto the final retentate vessel 8300 through a line 8132, the solutionfirst passing through a manual valve 8208 and a sterilizing hydrophilicfilter 8174, at a rate of 1.75 L per 0.50 L concentrated large diametersynthetic membrane vesicles suspension added to the final retentatevessel 8300. In some embodiments, the large diameter synthetic membranevesicles suspension can be pumped through the pump 8114 to thecross-flow filtration module 8154, wherein the permeate can be drawn offto be discarded through a permeate line 8164 (passing through asterilizing hydrophilic filter 8194 and a manual valve 8210) at a rateof 2.25 L per 0.5 L of large diameter synthetic membrane vesiclessuspension fed to the final product vessel 8300. In some embodiments, inthe final product vessel 8300, the large diameter synthetic membranevesicles suspension can contain a buffer concentration. In someembodiments, the buffer concentration can be as low as 2%.

Filters 8170, 8190, 8172, 8192, 8174, and 8194 are sterilizinghydrophilic filters. Filters 8180, 8182, and 8184 are sterilizinghydrophobic gas vent filters used in the retentate vessels and fed bygas lines 8131, 8133, and 8135, respectively. In some embodiments, thesystems depicted in FIG. 1A through FIG. 8 can be operated in a sterile(aseptic) fashion). For example, the addition of appropriate steamlines, condensate drain lines and valves, can allow the system to besterilized. In some embodiments, all inputs and outputs are equippedwith sterile barrier filters. In a typical embodiment, the largediameter synthetic membrane vesicles are multivesicular liposomes.

In some embodiments, a continuous-flow particle concentration system canbe configured where a set of cascading particle suspension concentratorscan be in series together adapted to remove and/or replace thesuspending medium of a particle suspension in a continuous fashion. Insome embodiments, the cascading particle suspension concentrators can becross flow filters (FIG. 8, components 8150, 8152, 8154) and thesuspending medium can be replaced by diafiltration. In some embodiments,the cascading particle suspension concentrators can be hydro-cyclones ordisk centrifuge units.

FIG. 9

FIG. 9A provides cross-sectional views of an atomized droplet 902, and alarge diameter synthetic membrane vesicles particle 912. In a typicalembodiment, the large diameter synthetic membrane vesicles particle 912can be formed by removal of an organic solvent from the atomized droplet902. In a typical embodiment, the atomized droplet 902 can comprise afirst component core and a buffer solution shell 904. In someembodiments, first component core can comprise a continuous phase 908and a suspension of droplets 906 in the continuous phase 908. In someembodiments, the droplets 906 can be aqueous phase droplets and can besurrounded by a continuous phase 908 that can be an organic solvent. Insome embodiments, the aqueous phase droplets can be from about 10 nm toabout 10μ in diameter. In some embodiments, the aqueous phase dropletscan be from about 500 nm to about 5μ in diameter. In a typicalembodiment, the aqueous phase droplets can be from about 100 nm to about2μ in diameter. For example the average diameter of the aqueous phasedroplets can be about 1μ in diameter. In some embodiments, the organicsolvent can then be removed in the solvent removal chamber to providedroplets of large diameter synthetic membrane vesicles 912 within ashell of dextrose/lysine 914. In a typical embodiment, the largediameter synthetic membrane vesicles droplet 912 are a multivesicularliposomes droplet. Multivesicular liposomes (MVL) are uniquely differentfrom other lipid-based drug delivery systems. Topologically, MVL aredefined as liposomes containing multiple non-concentric chambers 916within each droplet 912, resembling a “foam-like” matrix. The chambers916 of the MVL can have the same volume as the first componentparticles, (e.g. 1μ) as shown in FIG. 9A. The presence of internalmembranes distributed as a network throughout the MVL may serve toconfer increased mechanical strength to the vesicle, while stillmaintaining a high volume:lipid ratio. Thus, both structurally andfunctionally the MVL are unusual, novel and distinct from all othertypes of liposomes.

In one embodiment, a large diameter synthetic membrane vesicles droplet930 made by the instant processes can be, as depicted in FIG. 9B, forexample, an atomized droplet 922 containing equal volumes ofdextrose/lysine 924 and first component, where the diameter of theatomized droplet is approximately 39.7 μm. In a typical embodiment, thelarge diameter synthetic membrane vesicles droplet 930 can be amultivesicular liposomes droplet. In this embodiment, the firstcomponent core then is approximately 31.5 μm and the dextrose/lysineshell 924 can be approximately 4.1 μm thick. When the organic solvent isremoved from this atomized droplet 922, the resultant MVL 930 is 25 μmin diameter (the dextrose/lysine shell 924 is omitted for clarity). Insome embodiments, chambers 936 of the MVL 930 can have the same volumeas the suspension of droplets in the continuous phase of the firstcomponent.

FIG. 10 is a schematic of one example of a continuous-flow diafiltrationsystem 1010 comprising a continuous flow centrifuge. The large diametersynthetic membrane vesicles suspension can be pumped from a solventremoval vessel to the continuous-flow diafiltration system 1010.

In some embodiments, the continuous-flow diafiltration system 1010 caninclude a solvent removal vessel as depicted in FIG. 7 and as incomponent 70 of FIG. 1A and FIG. 1B. The large diameter syntheticmembrane vesicles suspension can travel to the diafiltration system 1010through a particle suspension inlet line 10120 (also seen in FIG. 7,component 7270), reaching a first retentate vessel 10100. In someembodiments, for each 1 L of large diameter synthetic membrane vesiclessuspension fed to the first retentate vessel 10100, a 2 L solution canbe fed into the first retentate vessel 10100 through a line 10130, thesolution first passing through a manual valve 10600 and a sterilizinghydrophilic filter 10170. In some embodiments, the large diametersynthetic membrane vesicles are multivesicular liposomes and thesolution is a saline solution.

In some embodiments, a portion of the large diameter synthetic membranevesicles suspension in the first retentate vessel 10100 can be pumped bypump 10110 through a cross-flow (tangential-flow) filtration module10150. For example, this can be a hollow fiber type module. In oneembodiment, the hollow fiber filter used is Model No. CFP-2-E-10A (0.2micron) manufactured by Amersham Biosciences of Westborough, Mass. Insome embodiments, the pore size of the cross-flow (tangential-flow)filtration modules 10150, 10152 and 10154, can be chosen to retain thelarge diameter synthetic membrane vesicles while allowing the suspendingmedium to pass through the filter membranes as permeate. The pumps usedin the diafiltration system can be of various types, such as peristalticor rotary lobe positive displacement pumps. In some embodiments,cross-flow recirculation pumps 10110, 10112 and 10114 operate with atleast twice the permeate flow rate of their associated cross-flow filtermodule and preferably 3 times, 5 times or 10 times the permeate flowrates. In some embodiments, the permeate can be drawn off through apermeate line 10160 (passing through a sterilizing hydrophilic filter10190 and a manual valve 10602), wherein for each 1 L of large diametersynthetic membrane vesicles suspension added to the first retentatevessel 10100, 2.25 L of permeate can be removed and discarded. In someembodiments, the retentate from the filtration module 10150 can becirculated back into the first retentate vessel 10100 via a retentateline 10140. In some embodiments, for each 1 L of large diametersynthetic membrane vesicles suspension added to the first retentatevessel 10100, a 0.75 L flow of the concentrated large diameter syntheticmembrane vesicles suspension can be removed from the first retentatevessel 10100 through a feed line 10122 and a metering pump 10123 to befurther filtered in a second retentate vessel 10200. In someembodiments, the large diameter synthetic membrane vesicles suspensionexiting the first retentate vessel 10100 can be concentrated by a factorof 1.33 (large diameter synthetic membrane vesicles concentration isincreased by 33%). In a typical embodiment, the large diameter syntheticmembrane vesicles are multivesicular liposomes.

In some embodiments, similar filtration can occur in the secondretentate vessel 10200 as described above for the first retentate vessel10100. In some embodiments, the large diameter synthetic membranevesicles suspension can enter the second retentate vessel 10200 at arate of 0.75 L per 1 L added to the first retentate vessel 10100. Insome embodiments, a solution can be fed into the second retentate vessel10200 through a line 10132, the solution first passing through a manualvalve 10204 and a sterilizing hydrophilic filter 10172, at a rate of 2 Lper 0.75 L concentrated large diameter synthetic membrane vesiclessuspension added to the second retentate vessel 10200. In someembodiments, a portion of the large diameter synthetic membrane vesiclessuspension in the second retentate vessel 10200 can be pumped by pump10112 through a cross-flow (tangential-flow) filtration module 10152. Insome embodiments, the permeate can be drawn off through a permeate line10162 (passing through a sterilizing hydrophilic filter 10192 and amanual valve 10606), wherein for each 0.75 L of large diameter syntheticmembrane vesicles suspension added to the second retentate vessel 10200,2.25 L of permeate can be removed and discarded. In some embodiments,the retentate from the filtration module 10152 can be circulated backinto the second retentate vessel 10200 via a retentate line 10142. Insome embodiments, for each 0.75 L of large diameter synthetic membranevesicles suspension added to the second retentate vessel 10200, a 0.50 Lflow of the concentrated large diameter synthetic membrane vesiclessuspension can be removed from the second retentate vessel 10200 througha feed line 10124 and a metering pump 10125 to be further filtered in athird retentate vessel 10300. In some embodiments, the large diametersynthetic membrane vesicles suspension exiting the second retentatevessel 10200 can now be concentrated 200%, whereas the buffer solutionconcentration in the large diameter synthetic membrane vesiclessuspension is roughly 9.1% with respect to the large diameter syntheticmembrane vesicles suspension in the inlet line 10120.

In some embodiments, similar filtration can occur in the third retentatevessel 10300 as described above for the first retentate vessel 10100 andsecond retentate vessel 10200. In some embodiments, the concentratedlarge diameter synthetic membrane vesicles suspension traveling throughthe feed line 10124 can enter the third product vessel 10300 where it isfurther filtered and concentrated to contain all the large diametersynthetic membrane vesicles entering through the inlet line 10120, inthe third product vessel 10300. In some embodiments, a solution can befed into the third retentate vessel 10300 through a line 10132, thesolution first passing through a manual valve 102010 and a sterilizinghydrophilic filter 10174, at a rate of 1.75 L per 0.50 L concentratedlarge diameter synthetic membrane vesicles suspension added to the thirdretentate vessel 10300. In some embodiments, the large diametersynthetic membrane vesicles suspension can be pumped through the pump10114 to the cross-flow filtration module 10154, wherein the permeatecan be drawn off to be discarded through a permeate line 10164 (passingthrough a sterilizing hydrophilic filter 10194 and a manual valve 10610)at a rate of 2.25 L per 0.5 L of large diameter synthetic membranevesicles suspension fed to the third product vessel 10300. In someembodiments, in the third product vessel 10300, the large diametersynthetic membrane vesicles suspension can contain a bufferconcentration. In some embodiments, the buffer concentration can be aslow as 2%.

In some embodiments, the large diameter synthetic membrane vesiclessuspension traveling through a feed line 10126, can be metered by a pump10127 and can enter a continuous flow centrifuge module 10400 whereinthe supernantant can be pumped to be discarded through a pump 10116through a permeate line 10166 (passing through a sterilizing hydrophilicfilter 10196). In a typical embodiment, the large diameter syntheticmembrane vesicles are multivesicular liposomes.

In some embodiments, the large diameter synthetic membrane vesiclessuspension traveling through a feed line 10128, can be metered by a pump10129 and can enter a final retentate vessel 10500. In a typicalembodiment, the large diameter synthetic membrane vesicles aremultivesicular liposomes.

Filters 10170, 10190, 10172, 10192, 10174, 10194, and 10196 aresterilizing hydrophilic filters. Filters 10180, 10182, 10184 and 10186are sterilizing hydrophobic gas vent filters used in the retentatevessels and fed by gas lines 10131, 10133, 10135 and 10137,respectively.

FIG. 11 is a schematic of one example of a continuous-flow centrifugesystem 1110 comprising a plurality of continuous flow centrifuges. Thelarge diameter synthetic membrane vesicles suspension can be pumped froma solvent removal vessel to the continuous-flow centrifuge system 1110.

In some embodiments, the continuous-flow centrifuge system 1110 caninclude the solvent removal vessel as depicted in FIG. 7. The largediameter synthetic membrane vesicles suspension can travel to thecentrifuge system 1110 through a particle suspension inlet line 11120(also seen in FIG. 7, component 7270, and as represented by components70 in FIG. 1a and FIG. 1B), reaching a first retentate vessel 11100. Insome embodiments, a large diameter synthetic membrane vesiclessuspension can be fed to the first retentate vessel 11100, and asolution can be fed into the first retentate vessel 11100 through a line11130, the solution first passing through a manual valve 11600 and asterilizing hydrophilic filter 11170. The large diameter syntheticmembrane vesicles suspension in the first retentate vessel 11100 can bepumped by a pump 11220 to a first centrifuge module 11150. In someembodiments, the permeate can be drawn off from the first centrifuge11150 through a permeate line 11160 (passing through a sterilizinghydrophilic filter 11190 being pumped by a pump 11110), and discarded.In some embodiments, large diameter synthetic membrane vesiclessuspension can exit the first centrifuge module 11150 through a feedline 11122, metered by a pump 11123, to be further processed in a secondretentate vessel 11200. In some embodiments, the large diametersynthetic membrane vesicles suspension flowing in feed line 11122 can beconcentrated by at least 33%. In a typical embodiment, the largediameter synthetic membrane vesicles are multivesicular liposomes andthe solution is a saline solution.

In some embodiments, the concentrated large diameter synthetic membranevesicles suspension can enter the second retentate vessel 11200 forfurther processing, and a solution can be fed through a line 11132, thesolution first passing through a manual valve 11604 and a sterilizinghydrophilic filter 11172. The large diameter synthetic membrane vesiclessuspension from the second retentate vessel 11200 can be pumped by apump 11240 to a second centrifuge module 11152. In some embodiments, thepermeate can be drawn off from the second centrifuge 11152 through apermeate line 11162 (passing through a sterilizing hydrophilic filter11192 being pumped by a pump 11120), and discarded. In some embodiments,large diameter synthetic membrane vesicles suspension can exit thesecond centrifuge module 11152 through a feed line 11124, metered by apump 11125, to be further processed in a third retentate vessel 11300.In some embodiments, the large diameter synthetic membrane vesiclessuspension flowing in feed line 11125 can be concentrated by at least33%. In a typical embodiment, the large diameter synthetic membranevesicles are multivesicular liposomes and the solution is a salinesolution.

In some embodiments, the large diameter synthetic membrane vesiclessuspension traveling through the feed line 11124, can be metered by apump 11125 and can enter the third retentate vessel 11300 where it canbe further processed. In some embodiments, a solution can be fed intothe third retentate vessel 11300 through a line 11134, the solutionfirst passing through a manual valve 11608 and a sterilizing hydrophilicfilter 11174. In some embodiments, the large diameter synthetic membranevesicles suspension can be pumped by a pump 11260 to a third centrifugemodule 11154. In some embodiments, the permeate can be drawn off fromthe centrifuge through a permeate line 11164 (passing through asterilizing hydrophilic filter 11194 being pumped by pump 11130), anddiscarded. In a typical embodiment, the large diameter syntheticmembrane vesicles are multivesicular liposomes.

In some embodiments, the concentrated large diameter synthetic membranevesicles suspension traveling through feed line 11166, can be metered bypump 11127 and can enter a final retentate vessel 11400. In a typicalembodiment, the large diameter synthetic membrane vesicles aremultivesicular liposomes.

Filters 11170, 11190, 11172, 11192, 11174, and 11194 are sterilizinghydrophilic filters. Filters 11180, 11182, 11184 and 11186 aresterilizing hydrophobic gas vent filters used in the retentate vesselsand fed by gas lines 11131, 11133, 11135 and 11137, respectively. In atypical embodiment, the large diameter synthetic membrane vesicles aremultivesicular liposomes.

The systems depicted in FIG. 1A through FIG. 11 can be operated in asterile (aseptic) fashion. With the addition of appropriate steam lines,condensate drain lines and valves, the system can be sterilized. Allinputs and outputs are equipped with sterile barrier filters.

In some embodiments, the large diameter synthetic membrane vesicles aremultivesicular liposomes. In some embodiments, the multivesicularliposomes further comprise bupivaciane, DEPC, DPPG, and tricaprylin. Insome embodiments, the multivesicular liposomes further comprisebupivacaine phosphate, DEPC, DPPG, and tricaprylin. In some embodiments,the multivesicular liposomes further comprise bupivacaine, DEPC, DPPG,tricaprylin and cholesterol. In some embodiments, the multivesicularliposomes further comprise bupivacaine phosphate, DEPC, DPPG,tricaprylin and cholesterol.

In some embodiments, the multivesicular liposomes further comprisebupivaciane, dextrose, L-Lysine, DEPC, DPPG, and tricaprylin. In someembodiments, the multivesicular liposomes further comprise bupivacainephosphate, dextrose, L-Lysine, DEPC, DPPG, and tricaprylin. In someembodiments, the multivesicular liposomes further comprise bupivacaine,dextrose, L-Lysine, DEPC, DPPG, tricaprylin and cholesterol. In someembodiments, the multivesicular liposomes further comprise bupivacainephosphate, dextrose, L-Lysine, DEPC, DPPG, tricaprylin and cholesterol.

In some embodiments, the multivesicular liposomes further comprisebupivaciane, dextrose, DEPC, DPPG, and tricaprylin. In some embodiments,the multivesicular liposomes further comprise bupivacaine phosphate,dextrose, DEPC, DPPG, and tricaprylin. In some embodiments, themultivesicular liposomes further comprise bupivacaine, dextrose, DEPC,DPPG, tricaprylin and cholesterol. In some embodiments, themultivesicular liposomes further comprise bupivacaine phosphate,dextrose, DEPC, DPPG, tricaprylin and cholesterol.

In some embodiments, the multivesicular liposomes further comprisebupivaciane, L-Lysine, DEPC, DPPG, and tricaprylin. In some embodiments,the multivesicular liposomes further comprise bupivacaine phosphate,L-Lysine, DEPC, DPPG, and tricaprylin. In some embodiments, themultivesicular liposomes further comprise bupivacaine, L-Lysine, DEPC,DPPG, tricaprylin and cholesterol. In some embodiments, themultivesicular liposomes further comprise bupivacaine phosphate,L-Lysine, DEPC, DPPG, tricaprylin and cholesterol.

In some embodiments, the multivesicular liposomes further comprisebupivacaine, morphine, cytarabine, or their pharmaceutically acceptablesalts as the therapeutic agent. In some embodiments, the multivesicularliposomes further comprise bupivacaine phosphate, morphine sulfate, orcytarabine HCl.

In another embodiment, any one of the above described embodiments can beused alone or in combination with any one or more of the above describedembodiments. For example, any above described atomizing nozzle,evaporation apparatus, continuous-flow emulsification system,continuous-flow diafiltration system, continuous-flow diafiltrationfurther comprising one or more centrifuges, continuous-flow centrifugesystem, or continuous processing system can be used alone or incombination. Thus, an evaporation apparatus can be used in conjunctionwith a three-fluid atomizing nozzle. This evaporation system/atomizingnozzle can be used with a continuous-flow emulsification system, asdepicted in FIGS. 1A, 1B, and 1C. The three-fluid atomizingnozzle/evaporation apparatus combination can be used in conjunction witha continuous-flow system, as depicted in FIGS. 8, 10, and 11. Any ofthese combinations can be used to make multivesicular liposomes. Inparticular any of the combinations can be used to make multivesicularliposomes containing bupivacaine or its salts as the therapeutic agent.

The following examples are meant to further illustrate the embodiments,they are not meant to be limiting in any way.

Example 1

The following is an example utilizing the process parameters and stepsof the devices depicted in the Figures. The three fluids applied to theatomizing nozzle (FIG. 1A and FIG. 1B, component 75; FIG. 3A, component310; FIG. 7, component 7510) as part of the process of formingmultivesicular liposomes have the following compositions per liter.

The first fluid (FIG. 3A-3L, component 3115; FIG. 5, component 5115;FIG. 7, component 7115) was a first liquid made up of the firstcomponent, the first component having two components: an organic phaseand a first aqueous phase which are emulsified with equal volumes. Theorganic phase was composed of 1,2-dierucoyl-sn-glycero-3-phosphocholine(17.78 g), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (1.056 g),cholesterol (10.34 g), tricaprylin (4.32 g), water (0.70 g) andmethylene chloride (quantity sufficient to make 1 L total volume of theorganic phase. The first aqueous phase was composed of 0.2 molar (200mM) phosphoric acid and bupivacaine (40 g) and water (quantitysufficient to make 1 L total volume of the first aqueous phase).

The second fluid (FIG. 3A-3L, component 3120; FIG. 5, component 5120;FIG. 7, component 7120) was a second liquid made up of a second aqueousphase composed of L-lysine (monohydrate) (16.8 g), dextrose (13.25 g),and water (quantity sufficient to make 1 L total volume of the secondfluid.

The third fluid (FIG. 3A-3L, component 3140; FIG. 5, component 5140;FIG. 7, component 7140) was nitrogen gas containing water vapor (100%relative humidity at 42° C. and 25 psig).

Preparation of Solvent Evaporation Chamber

Nitrogen was supplied to the solvent removal vessel (FIG. 1A AND FIG.1B, component 50; FIG. 7 component 710) through two solvent removalvessel gas inlet lines: FIG. 1A components 115 and 110; FIG. 7components 7280 and 7430) the main carrier gas inlet line (component 115of FIG. 1A AND FIG. 1B; component 7280 of FIG. 7), or main rotation jet,was tangential to vessel wall and approximately 40% up from vesselbottom, causing clockwise rotation viewed from above; and the lidprotection gas inlet (component 110 of FIG. 1A AND FIG. 1B; component7430 of FIG. 7), or lid protection jet, at the corner of lid (component7220 of FIG. 7) and vessel wall (component 7350 of FIG. 7), tangentialto the wall and moving in the same rotational direction (component 7240of FIG. 7). The nitrogen entering these inlet lines was humidified to100% relative humidity at 42° C. and at 25 psig (pounds per square inchgauge). The main rotation jet supplies 335 L/min at 42° C. of humidifiednitrogen while the lid protection jet flow was 25 L/min at 42° C. ofhumidified nitrogen (the lid protection jet keeps deposit buildup offthe lid). The nitrogen was humidified prior to entering the solventremoval vessel by passing it through a heated tube-in-shell heatexchanger (component 90, FIG. 1A AND FIG. 1B) that was coated with water(humidification water) followed by an excess-liquid water removalchamber (component 45, FIG. 1A AND FIG. 1B) and liquid bleed. Thenitrogen was humidified to reduce evaporation of water from the spraywhich raised the osmolality of the suspending buffer.

In this example, the solvent removal vessel had a volume ofapproximately 138 liters, the inside diameter was 56 cm, the walls were52 cm high and the dome at the bottom was 10 cm deep. Thus the insideheight of the solvent removal vessel was 62 cm from the lid to thebottom of the domed bottom. The gas outlet tube (component 80, FIG. 1AAND FIG. 1B; component 7340, FIG. 7) (including the conical fitting FIG.7, component 300 and vortex stabilizer FIG. 7, component 7360) extends42.5 cm into the solvent removal vessel down from lid. The diameter ofthe gas outlet tube was 2.3 cm (inside diameter) and the conical fittingtapers 20 degrees to a 1.5 cm inside diameter for the gas outlet orifice(FIG. 7, component 7310). The vortex stabilizer attached to the end ofthe conical fitting had a diameter of 2.5 cm. The main rotation(carrier) gas inlet was tangential to the wall and 37 cm down from thelid and had an inside diameter of 1.9 cm.

The three-fluid atomizing nozzle (FIG. 1A and FIG. 1B, component 75;FIG. 3A, component 310; FIG. 7, component 7510), the rinse nozzle (FIG.1A AND FIG. 1B, component 105; FIG. 7, component 7400), and lidprotection gas inlet (FIG. 1A, component 110; FIG. 7, component 7340)all extend through the lid of the solvent removal vessel (FIG. 1A ANDFIG. 1B, component 50; FIG. 7 component 710) and were each centered 10.2cm from the vessel wall (FIG. 7, component 7350). The lid protection gasinlet (FIG. 7, component 7430) had an inside diameter of 0.95 cm,extends through the lid of the vessel (FIG. 7, component 7220), andfaced parallel to the inside of the lid in a direction to give clockwise(viewed down from above) rotation of the gas. Starting with thethree-fluid atomizing nozzle (FIG. 1A and FIG. 1B, component 75; FIG.3A, component 310; FIG. 7, component 7510) as zero degrees, the maincarrier gas inlet line was located 52 degrees clockwise, the rinsenozzle was 90 degrees clockwise, and the lid protection gas inlet was135 degrees clockwise with its outlet approximately 180 degreesclockwise.

The sides (FIG. 7, component 7350) and bottom (FIG. 7, component 7250)of the solvent removal vessel were jacketed at 24.1° C. by connection toa circulating bath. The temperature in the jacket was adjusted toapproximately match the steady state gas exit temperature. Such a matchprevented evaporation and drying of the multivesicular liposomes on thewall or condensation of water that could rupture, through osmotic shock,the multivesicular liposomes that are being formed in the vessel.

Preparation of a First Component

The recirculation loop (FIG. 2, component 2125) connected to thehigh-shear mixer (FIG. 1A AND FIG. 1B, component 25, FIG. 2, component2130) (Ross model HSM-703XS-20 Sanitary Inline High Shear Mixer equippedwith a 3″ diameter X-5 Series rotor/stator for operation to 14,400 rpm.(11,300 feet/min. tip speed) with gap ring #3) was primed with methylenechloride to ensure that all air was removed from the high-shear mixer.The jacket of the heat exchanger (FIG. 1A AND FIG. 1B, component 30;FIG. 2 component 2170) was supplied with 5° C. coolant (water+50%ethylene glycol) (FIG. 1A, components 96 and 97; FIG. 2, components 2110and 2105). The mixer seal lubricant tank (FIG. 1A AND FIG. 1B, component10), filled with water, was also cooled with 5° C. coolant (water+50%ethylene glycol). The mixer was started at a setting of 30 Hz (approx.7,200 rpm) causing a flow around the mixer loop estimated at 21,000mL/min and with an internal volume of 280 mL. Thus, fluid in the loopwent through the mixer blades and heat exchanger an average of every 0.8seconds.

After the high shear mixer was primed with methylene chloride, theorganic phase and first aqueous phase peristaltic pumps (components 12and 2, respectively, of FIG. 1A AND FIG. 1B) were concurrently started.The organic phase was pumped at 33 mL/min and the first aqueous phasewas also pumped at 33 mL/min. The organic phase and first aqueous phaseentered the high-shear mixer starting the formation of the firstcomponent. As the first component circulated around the high shear mixerrecirculation loop, a small fraction of the flow was forced through thefirst component exit line (FIG. 2, component 2180; FIG. 5, component5114) to the three-fluid atomizing nozzle (FIG. 1A AND FIG. 1B,component 75; FIG. 3A-3L, component 310; FIG. 5, component 505; FIG. 7,component 7510) at 66 mL/min (total of the two flow rates).Consequently, the priming methylene chloride was soon flushed from themixer loop by this flow (4.2 min per loop volume of flush).

Concurrent with the starting of the organic phase and first aqueousphase peristaltic pumps, the second aqueous phase (connected to thethree-fluid atomizing nozzle) and wall rinse (to the rinse nozzle (FIG.1A and FIG. 1B, component 105; FIG. 7, component 7400)) peristalticpumps (components 22 and 64, respectively, of FIG. 1A and FIG. 1B) werestarted and pumped their respective components at 66 mL/min each. Thewall rinse solution was 33.5 g of dextrose per liter of water. Nitrogenat 60 psig (room temperature, not humidified) was supplied to these twonozzles (FIG. 1A and FIG. 1B, components 75 and 105; FIG. 7, components7510 and 7400). The second aqueous phase flowed through the three fluidnozzle at 66 mL/min. The three-fluid atomizing nozzle had a nitrogenflow rate of 51 L/min @ 1 atm and the wall rinse nozzle (manufactured byGEA Process Engineering of Columbia, Md.) had a flow rate of 66 L/min @1 atm.

After exiting the three-fluid atomizing nozzle, the formed atomizedemulsion droplets came into contact with the carrier gas (nitrogen) inthe solvent removal chamber (FIG. 7, component 7230) of the solventremoval vessel (FIG. 1A and FIG. 1B, component 50; (FIG. 7, component710). The carrier gas rotated inside the chamber (FIG. 7, component7240) as a small, rapidly rotating, intense gas vortex (FIG. 7,component 7245) formed at the exit orifice (FIG. 7, component 7310).This allowed the droplets to contact the gas for an extended period oftime in order to effectuate methylene chloride evaporation and removal.After removal of the methylene chloride, the formed multivesicularliposome suspension droplets (FIG. 7, component 7380) were collected asa suspension of multivesicular liposomes (FIG. 7, component 7390) at thebottom of an evaporation vessel (FIG. 1A and FIG. 1B, component 50; FIG.7, component 7250). The multivesicular liposomes were collected in thesolvent removal vessel and then drained out of the drain port (FIG. 1Aand FIG. 1B, component 130; FIG. 7, component 7270) through aperistaltic positive displacement pump (FIG. 1A and FIG. 1B, component125), set to pump slightly faster (approximately 200 mL/min) than thesuspension drains out of the bottom (approximately 165 mL/min),therefore the exit stream of multivesicular liposome suspension wereperiodically interrupted by small segments of chamber gas. This pumprate prevented any appreciable venting of solvent vapors into the roomand protects the multivesicular liposome suspension from exposure tohigh velocity gas streams or foaming.

At system equilibrium, the nitrogen exiting the solvent removal vesselthrough the gas outlet (FIG. 1A and FIG. 1B, component 80; FIG. 7,component 7310) was at a temperature of approximately 21.5° C. Thesolvent removal vessel jacket was cooled to approximately 24.1° C. Thetemperature of the first component (part of which travels to thethree-fluid atomizing nozzle) leaving the heat-exchanger was 15.3° C.After traveling through heat exchanger, and while traveling back to thehigh-shear mixer through the recirculation line, the temperature of thefirst component was approximately 14.3° C.

At equilibrium the system then ran continuously making multivesicularliposomes for as long as the feed solutions and nitrogen were supplied.Five hundred mL samples of multivesicular liposome suspension were takenfrom the suspension outlet (FIG. 7, component 7260) in the solventremoval vessel and diafiltered (using a hollow fiber filter, Model No.CFP-2-E-8A from Amersham Biosciences of Westborough, Mass.) with fourvolumes of normal saline on a small scale batch diafiltration system(FIG. 1A and FIG. 1B, component 70). Optionally, settling of themultivesicular liposomes and excess liquid could be decanted to obtainthe final multivesicular liposomes in a chosen aqueous solution at achosen MVL concentration. Full continuous operation can be obtained byconnecting the solvent removal vessel outlet pump to the apparatus ofFIG. 8.

Example 2

Preparation of First Component

The recirculation loop connected to the high-shear mixer (FIG. 1A andFIG. 1B, component 25; FIG. 2, component 2130) (Ross Model HSM-703XS-20Sanitary Inline High Shear Mixer equipped with a 3″ diameter X-5 Seriesrotor/stator for operation to 14,400 rpm. (11,300 feet/min. tip speed)with gap ring #3) was primed with methylene chloride to ensure that allair was removed from the high-shear mixer. The jacket of the heatexchanger (FIG. 1A and FIG. 1B, component 30; FIG. 2, component 2170)was supplied with 5° C. coolant (water+50% ethylene glycol). The mixerseal lubricant tank, filled with water, was also cooled with 5° C.coolant (water+50% ethylene glycol). The high-shear mixer was started ata setting of 25 Hz (6,000 rpm), 30 Hz approx. (7,200 rpm) or 35 Hz(8,400 rpm).

After the high shear mixer was primed with methylene chloride, theorganic phase and first aqueous phase peristaltic pumps (FIG. 1A andFIG. 1B, components 12 and 2, respectively) were concurrently started.The organic phase was pumped at 33 mL/min and the first aqueous phasewas also pumped at 33 mL/min. Entry of the organic phase and firstaqueous phase begin formation of the first component. As the firstcomponent circulates around the high shear mixer recirculation loop(FIG. 2, component 2125), a small fraction of the flow was forcedthrough the first component exit line (FIG. 2, component 2180) to thethree-fluid atomizing nozzle (FIG. 1A and FIG. 1B, component 75; FIG.3A; FIG. 4A; FIG. 77510) at 66 mL/min (total of the two flow rates), thetemperature of the emulsion being forced through the first componentexit line (FIG. 2, component 2180) was from 16.5 to 21.1° C. The flow ofthe organic phase and first aqueous rapidly flushed the primingmethylene chloride from the mixer loop (4.2 min per loop volume offlush).

TABLE 1 Organic Phase Components (per 2 L Total Volume) cholesterol(Nippon) 20.8 g DEPC (Nippon) 36.0 g DPPG (Lipoid) 1.89 g tricaprylin(NOF) 8.81 g Water for injection 0.49 mL Methylene chloride (EMD) 2,569g

The components of the organic phase are shown in Table 1.

TABLE 2 First Aqueous Phase Components (per 2 L Total Volume)Bupivacaine (BASF)  80 g 0.20M H₃PO₄ (2 L, Mallinckrodt) 200 mM

The components of the first aqueous phase are shown in Table 2.

TABLE 3 Particle Sizing of Diluted Emulsion Samples by laser lightscatter analysis. PSD (μm)(volume based) Batch d₁₀ d₅₀ d₉₀ Span Ross 30Hz 0.5 0.9 1.4 1.0 Ross 35 Hz 0.5 0.8 1.3 1.0 Ross 25 Hz 0.6 1.1 1.7 1.0Batch Lot D 0.855 1.151 1.506 0.566 Batch Lot E 0.720 1.110 1.530 0.730

The emulsion samples were diluted and analyzed using a light scatteringdevice (Horiba Instruments La-910) and the results are shown in Table 1.

The second aqueous phase (connected to the three-fluid atomizing nozzle)and wall rinse (to the rinse nozzle (FIG. 1A and FIG. 1B, component 105;FIG. 7, component 7400)) peristaltic pumps (components 22 and 64,respectively, of FIG. 1A and FIG. 1B) were started when the organicphase and first aqueous phase peristaltic pumps were started. The wallrinse solution having 33.5 g of dextrose per liter of water wasintroduced to the evaporation chamber at a flow rate of 66 mL/min.Nitrogen at 60 psig (room temperature, not humidified) was supplied tothe atomizing nozzle. The second aqueous phase was introduced to thethree fluid nozzle at a flow rate of 66 mL/min. The three-fluidatomizing nozzle has a nitrogen flow rate of 51 L/min @ 1 atm and thewall rinse nozzle (manufactured by GEA Process Engineering of Columbia,Md.) has a flow rate of 66 L/min @ 1 atm. The emulsion, second aqueousphase and nitrogen gas were combined using the three fluid nozzle toafford atomized droplets (FIG. 3, component 3155; FIG. 7, component7155) which traveled in the evaporation chamber until the majority ofthe methylene chloride was removed from the atomized droplets. Removalof the methylene chloride produced multivesicular liposomes (FIG. 7,component 7380) which form a suspension (FIG. 7, component 7390) at thebottom of the evaporation chamber (FIG. 7, component 7250).

TABLE 4 Second Aqueous Phase Components (per 5 L Total Volume) L-Lysinemonohydrate 84 g 50% Dextrose soln (B Braun) 132.5 mL Deionized water(to final volume) 5 L The components of the second aqueous phase areshown in Table 4.

After system equilibrated (10 minutes), 500 mL samples of multivesicularliposomes suspension were collected. The Ross RPM was then changed andallowed to equilibrate for 10 minutes before collecting the next 500 mLsample. This was repeated for the last RPM. Each 500 mL batch wasdiafiltered in a batch mode (using a hollow fiber filter, Model No.CFP-6-D-9A from Amersham Biosciences of Westborough, Mass.), for fourvolumes with normal saline on a small scale batch diafiltration system(FIG. 1, component 70). The recirculation pump was set to 5,700 ml/min,while saline addition pump was set to 410 mL/min. The permeate valve wasadjusted to keep the liquid volume in the diafiltration system constantat 1,000 and thus was also 410 mL/min, since the minimum working volumeof this system was approximately 550 mL. In the present example, theprocessed multivesicular liposomes suspension was allowed to settle at5° C. and then supernatant was decanted to achieve approximately 15 mgof bupivacaine per mL in the final multivesicular liposomes suspension.

TABLE 5 Wall Rinse Solution (per 5 L Total Volume) 50% Dextrose soln (BBraun) 335 mL Deionized water (to final volume)  5 L

As can be seen in FIG. 12, controlled release of bupivacaine from MVLparticles was examined in vitro at 37° C. in 0.5% ovine serum albumindissolved in 50 mM phosphate buffered saline (pH7) showing a similarrelease of bupivacaine to that in Batch Lot A and B made by the processdisclosed in patent WO 99/25319.

As can be seen in FIG. 13, the PK profile in rats of the continuousprocess samples shows a similar sustained release profile of bupivacaineto that in Batch Lot C made by the batch process disclosed in patent WO99/25319.

TABLE 6 Final Material Properties Total Free Bupi^(i) % Bupi^(i) %PSD^(iii) (μm) pH Total Lipids Batch (mg/mL) PPV^(ii) (mg/mL) Free d10d50 d90 Int Ext Chol^(iv) DEPC^(v) DPPG^(vi) TC^(vii) 30 Hz 14.9 41 0.793.2 18.0 44.7 102.8 5.7 7.0 3.24 5.98 0.23 1.50 Batch 35 Hz 13.5 38 0.813.7 14.2 36.3  83.2 6.0 7.3 3.70 6.72 0.31 1.61 Batch 25 Hz 12.8 38 0.834.0 14.8 32.4  69.4 5.9 7.4 3.30 6.15 0.22 1.51 Batch ^(i)bupivacaine;^(ii)packed particle volume; ^(iii)particle size distribution by mass;^(iv)cholesterol; ^(v)1,2-dierucoyl-sn-glycero-3-phosphocholine;^(vi)1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol; ^(vii)tricaprylin

Example 3

Heat Treatment of MVL Suspension

The system of FIG. 1B was used with the humidified rotation gas (N2)supplied by combination electric heater and tube-in shell heat exchangeras described for FIG. 1A, component 90. The system was equilibrated for10 minutes and a 1,000 ml sample of MVL suspension, exiting the drainport (FIG. 1B, component 130) of the solvent removal vessel 50, wascollected. The MVL sample was divided into two samples of 500 mL each.The first 500 mL MVL sample was heat treated as follows. The heattreatment was performed by rapidly adding 750 mL of 100° C. dextrosesolution to the first sample to raise the mixture temperature up toapproximately 63° C. After 30 seconds, 1,750 mL of +5° C. saline wasrapidly added to lower the temperature of the mixture to near roomtemperature (35° C. or below). The sample volume was now 3,000 mL. Thesecond 500 mL multivesicular liposomes sample was not heat treated. Thesecond sample was diluted with the same volumes of dextrose solution(750 mL) and saline (1,750 mL) as the first sample but the solutionswere at room temperature.

Each sample was diafiltered batch wise, with 4 volumes of saline. These3,000 ml samples were each concentrated to 1 liter and then diafilteredin a batch mode (using a hollow fiber filter, Model No. CFP-6-D-9A fromAmersham Biosciences of Westborough, Mass.), for four volumes withnormal saline on a small scale batch diafiltration system (FIG. 1B,component 70). The recirculation pump (FIG. 8, component 8110, 8112, and8114) was set to 5,700 mL/min, while saline addition pump (FIG. 8,component 8170, 8172, and 8174) was set to 410 mL/min. The permeatevalve (FIG. 8, component 8202, 8206, and 8210) was adjusted to 410mL/min to keep the liquid volume in the system constant at 1,000 mL.Since the minimum working volume of this system was approximately 550ml, which was too large to allow these small samples to be concentratedto the target bupivacaine concentration of 15 mg/mL, they were allowedto settle at +5° C. and the supernatant was decanted to achieveapproximately 15 mg of bupivacaine per ml in the final MVL suspensions.The analytical results for the samples were as follows:

TABLE 7 d₁₀ d₅₀ d₉₀ ppv bupi/ml Not heat treated 14.9 52.8 107.7 56%16.84 Heat treated 18.2 51.3 95.9 55% 18

TABLE 8 Chol DEPC DPPG Tricap Sample Total mg/ml Not heat treated 4.978.50 0.39 2.11 Heat treated 7.07 12.35 0.58 3.00

As can be seen in the Tables 7 and 8 above, the heat treatment did notsignificantly effect the particle size distribution of the MVLs and hadonly small effects on the bupivacaine (active agent) content and lipidcomposition of the particles.

The heat treatment did have a surprising effect on the acceleratedstability (30° C. stability) of these MVL suspensions, as can be seen inFIG. 14. Accelerated stability at 30° C. was greatly improved with heattreatment. A lower slope means less bupivacaine release and longerstability. Both samples were usable products when stored at +5° C. butthe heat treated sample was projected to have a much longer shelf life.

As can be seen in FIG. 15, The PK profile in rats (measuredsubstantially as per patent number WO 02/096368) also shows animprovement with heat treatment. Both samples have acceptable PKprofiles. The heat treated sample gives longer lasting sustained releasewith higher serum values at 48 and 72 hours. The not heat treated samplewas essentially zero at 72 hours. The heat treatment surprisinglyimproved the accelerated stability of the MVLs and also improved theirin-vivo release profile.

Example 4

Effect of Lowered 1st Aqueous Osmolality

The osmolality of the 1st aqueous solution in the previous examples wassignificantly above 300 mOsm/kg. If there was no appreciable loss ofbupivacaine or phosphoric acid or water transport across the formingphospholipid membranes during the MVL production process, the internalchambers of the resultant MVLs will be filled with an aqueous solutionwith an osmolality at or near that of the final saline (300 mOsm/kg)storage suspending medium. If, on the other hand, the osmolality of the1st aqueous and resultant MVLs was lower than that of saline, the MVLswill shrink slightly as the saline draws water out of the internalchambers. This will compress the phospholipids making up the MVLmembranes and make them more stable and less bupivacaine permeable.

Both the lipid-solvent (LC) and the bupivacaine-acid (1st aqueous) weremade up at ½ the concentration of normal (see below for formulations).This gave a 1st aqueous solution with an osmolality of 189 mOsm/kg whichwas expected to cause the MVLs to shrink and compress their membraneswhen diafiltered into normal saline of 300 mOsm/kg.

TABLE 9 FIG. 2 #s FIG. 1B #s (but with no sterile filters) 2160 12 LC,Solvent-lipids pump 33 ml/min 2120 2 1st aqueous, acid Bupi pump 33ml/min 22 Dextrose-lysine pump, 3 fluid noz 66 ml/min 64 Dextrose onlypump, wall rinse noz 66 ml/min 120 Output rate out of spray chamber 165ml/min 115 & 110 Temperature of water humidified N2 44.6 C. feedingrotation jets 90 Temperature of N2 exiting electric heater 46 C 90 40Flow rate of humidification water into 9 ml/min steam generator 33 Flowto both rotation jets (N2 before 400 L/min steam) 57 Flow to toprotation jet 15 L/min Chamber Jacket supply temperature 25 C. Chamberexit temperature 21.9 C. 2150 & 2180 heat exchanger emulsion inletsupplied to 18.8 C. 3 fluid nozzle 2175 heat exchanger emulsion outlet17.2 C. 2110 Heat exchanger coolant supply 4 C. 11 3 Fluid nozzle N2pressure 60 psig 21 Wall rinse nozzle pressure 60 psig 13 3 fluid nozzleN2 flow 54 L/min 23 Wall rinse nozzle N2 flow 53 L/min 25 Ross Mixeralways with gap ring #3 Hz on Run at 30 VFD resulting in this bladerotation rate 7,200 RPM 2160 10 Solvent solution (LC), 4 liters 4 literscholesterol Nippon) 20.8 g DEPC (Nippon) 36.0 g DPPG (Lipoid) 1.89 gtricaprylin (NOF) 8.81 g WFI 1.4 ml MeCl (EMD) 5,138 g 2120 5 1staqueous soln. Acid & Bupi, 2 liters 2 liters (emulsified by Ross, fed torotor center) Dissolve Bupivacaine base (BASF) 40 g in 2 liters of0.112M H3PO4 (Mallinckrodt) 112 mM Osmolality 189 mOsm 60Dextrose/lysine solution 5 liters (fed to sheath of 3 fluid nozzle)L-Lysine monohydrate 84 g 50% Dextrose soln (B Braun) 105 g DI water tofinal volume of 5 L Osmolality 145 mOsm 66 Dextrose wall rinse solution5 liters 50% Dextrose soln (B Braun) 329 g DI water to final volume of 5L Osmolality 148 mOsm

Since only ½ the lipids and ½ the previous examples bupivacaine areprocessed per minute but the Dextrose Lysine (second aqueous) andDextrose only (wall rinse) solutions are pumped at the normal rate, theconcentration of bupivacaine MVLs out of the spray chamber (FIG. 1,component 50) will be ½ of that seen in the previous examples. A larger1,000 mL sample was therefore taken for concentration and diafiltrationas in the previous example. This sample was not heat treated.

TABLE 10 Analysis pf the final decanted product MVLs are as follows:Bupi mg/ml % free PPV % d10 d50 D90 15.21 1.1% 71% 9.3 22.5 55.5

The accelerated stability (30 C) plot for this low osmolality sample canbe seen in FIG. 14. The low osmolality sample was significantly morestable than the not heat treated sample but less stable than the heattreated sample.

As seen in FIG. 15, the Rat in-vivo release profile of the lowosmolality sample can be the most desirable as it has the lowest initialpeak and the longest duration; highest blood concentrations at 72 and 96hours. In this process, starting with a 1^(st) aqueous solution with anosmolality lower than the final MVL suspending solution was unexpectedlyfound to both increase MVL storage stability, reduce the initial in-vivorelease peak and prolong the duration of bupivacaine delivery.

As can be seen from Table 10, this example produced a smaller particlesize d50 than the previous examples. As can be seen from FIG. 14, thelow osmolality sample, which was not heat treated, has a much betteraccelerated stability than the not heat treated sample of the previousexample.

Example 5

Solvent Exchange/Concentration

FIGS. 8, 10 and 11 depict example continuous buffer exchange and MVLconcentration systems (seen in FIG. 1A and FIG. 1B, component 70) whichare fed a MVL suspension from a solvent removal vessel (seen in FIG. 1Aand FIG. 1B, component 50) by a solvent line (seen in FIG. 1A and FIG.1B, component 120).

These systems take the continuous flow of MVL suspension produced in asolvent removal vessel (seen in FIG. 1A and FIG. 1B, component 50) andexchange the suspending buffer for another suspending medium (e.g.normal saline) and at the same time optionally concentrate the MVLsuspension. In most cases it is desired to produce slightly overconcentrated MVL suspensions in a retentate vessel (seen in FIG. 8,component 8300; FIG. 10, component 10500; and FIG. 11, component 11400).The lot is analyzed and sterilely diluted to exact concentration beforefilling into vials aseptically. The final concentration of the MVL isobtained by processing in two or more stages connected in series.Additionally, processing results in any percent of original suspendingbuffer removal.

These systems include medium supply source, such as a tank and aparticle concentrating device. The particle concentrating device is ahollow fiber tangential flow filter (e.g. Model No. CFP-6-D-9A fromAmersham Biosciences of Westborough, Mass.) or a continuous orsemi-continuous centrifuge (Centritech Lab III or CARR ViaFuge Pilotfrom Pneumatic Scale Angelus Corp., Clearwater, Fla.) or any otherdevice that separates the MVLs from the suspending medium.

The following constraints apply to analogous components of all thesteady state stages of the continuous buffer exchange and MVLconcentration systems:

In a continuous manner, the first stage of FIG. 8 consisting ofcomponents 8120, 8100, 8122, 8123, 8110, 8190, 8202, 8160, 8150, 8140,8180, 8131, 8130, 8212 and 8170 will exchange the initial suspendingbuffer with normal saline.

In reference to FIG. 8, at steady state where a constant volume is inthe tank, the input volume flows; MVL suspension in 8120 and saline in8130 must equal the volume out flows; permeate 8160 and MVL suspensiontransferred to the next stage by metering pump 8123. This is done bycontrolling/fine adjusting any of the above 4 flow rates to keep thevolume in the tank constant, e.g. saline fine adjustment valve 8212.

At steady state the mass or number of MVLs is conserved and thus theinput rate of the number of MVLs, concentration times the flow rate,from pipe 8120 must equal the MVL outflow from 8122, again numberconcentration times flow rate. This means that if the volume outflowrate in 8122 is lower than the input volume flow rate in 8120, theconcentration of MVLs in the tank 8100 and pipe 8122 rises until the MVLin matches the MVL out. This gives a MVL concentration factor, outputMVL concentration divided by the input MVL concentration equal to thevolume flow in MVL input pipe, 8120, divided by the volume flow in theoutput pipe, 8122.

The original buffer is diluted by saline at each stage. This dilutionfactor is the flow rate of buffer in to the tank, 8100, divided by thetotal of the volume flow rate of buffer in pipe 8120 and the volume flowrate of saline in pipe 8212. The MVLs take up appreciable volume and sothe flow rate of buffer into the tank, 8100, is the volume flow rate inpipe 8120 times one minus the volume percentage of that suspension thatis MVLs, (PPV %, Packed Particle Volume percentage).

Adhering to these constraints, the first 2 stages of FIG. 8, containingtanks 8100 and 8200 will reach steady state. The smaller the tanks, thefaster the equilibration time and the lower the total holdup in thesystem. For the systems described in Examples 2 and 3, a tank volume offrom 0.25 liter to 10 liters and preferably 0.75 liters is appropriate.

The third stage in FIG. 8 contains the final retentate vessel, 8300, andis large enough to hold one lot of product, e.g. 40 liters. As shown,the volume in final retentate vessel 8300 remains constant but theconcentration is continuously rising as there is no MVL out flow, untilit reaches the desired final concentration of MVLs. By choosingappropriate flow rates in pipes 8124, 8208 and 8164 this stage isalternatively run with a constant MVL concentration and rising volume.

When starting these systems, the systems are either started with eachtank filled to the appropriate volume with saline or they are startedempty and filled with the MVL suspension input and saline input but onlystart the output pump, e.g. 8123, and hollow fiber recirculation pump,e.g. 8110 when the tank for that stage fills to its desired volume.Likewise at the end of a lot, the MVL input line is switched to salinewhich moves all MVLs to the product tank, or the saline and MVL inputsand the hollow fiber recirculation pump is stopped to allow each tank toempty into the next until the lot is again all in the product vessel.

Flow rate and additional parameters for the system of FIG. 8 using thehollow fiber cartridge and filtration rates and MVL output rate of the25 Hz Process, 30 Hz Process, or 35 Hz Process of Example 1 is givenbelow:

TABLE 11 (flow rates are for fluid traveling through the components fromFIG. 8) concentration factor of 100% in 1st 2 stages (buffer exchangeonly) MVL suspension in Saline in MVL suspension out permeate Stage(mL/min) (mL/min) (mL/min) (mL/min) 1 8120 165 8130 410 8122 165 8160410 2 8122 165 8132 410 8124 165 8162 410 3 8124 165 8134 245 0 8164 410Lysine Net tank volume part conc. dilution % Lysine % Stage change outPPV % factor left exchanged System 810 Input, 100% 20.0% 100%  8120 >>>1 8100 0 100% 20.0% 0.2435 24.4%  75.6% 2 8200 0 100% 20.0% 0.2435 5.9%94.1% 3 8300 0 rising rising 0.3501 2.1% 97.9%

The flow rates in Table 11 result in buffer exchange only. The MVL PPVremains at 20% but the original buffer concentration is reduced by 99.7%with its concentration in the product being 2.1% of original. Forcomparison, a 4 volume batch diafiltration exchange as used in theexamples, reduces the buffer concentration to 1.8% or 98.2% exchanged.

TABLE 12 (flow rates are for fluid traveling through the components fromFIG. 8) A total concentration factor of 200% in 1st 2 stages and bufferexchange MVL suspension in Saline in MVL suspension out permeate Stage(mL/min) (mL/min) (mL/min) (mL/min) 1 8120 165 8130 369 8122 124 8160410 2 8122 124 8132 396 8124 110 8162 410 3 8124 110 8134 300 0 8164 410Stage Lysine Net tank volume concentration dilution % Lysine % Stagechange factor PPV % factor left exchanged System 810 Input, 100% 20.0%100%  8120 >>> 1 8100 0 133% 26.6% 0.2635 26.3%  73.7% 2 8200 0 150%40.0% 0.1870 4.9% 95.1% 3 8300 0 rising rising 0.1798 0.9% 99.1%

The flow rates in Table 12 result in buffer 99.1% exchanged down to 0.9%of the original concentration.

TABLE 13 (flow rates are for fluid traveling through the components fromFIG. 11) concentration factor of 300% in 3 stages and buffer exchangeProduct is collected in Tank 11400 with volume rising at 114.3 mL/minMVL suspension in Saline MVL suspension out Supernatant out Stage(mL/min) (mL/min) (mL/min) (ml/min) 1 11120 165 11130 949 11122 114.311160 1,000 2 11122 114 11132 1000 11124 114.3 11162 1,000 3 11124 11411134 1000 11166 114.3 11164 1,000 Stage Lysine Net tank volumeconcentration dilution % Lysine % Stage change factor PPV % factor leftexchanged System 1110 Input, 100% 20.0% 100%  11120 >>> 1 11100 0 144%28.9% 0.1221 12.2%  87.8% 2 11200 0 144% 41.7% 0.0752 0.9% 99.1% 3 11300 144% 60.1% 0.0625 0.1% 99.9%

The flow rates in Table 13 result in a concentration factor of 300% withthe same input conditions used for Tables 11 and 12.

Systems can be assembled with any combination of hollow fiber cartridgesand centrifuges. They can have two or three or more stages. With morestages the buffer is exchanged with less saline but there is moreequipment to keep aseptic.

The semi-continuous centrifuges above, Centritech Lab III or CARRViaFuge Pilot, are operated in an aseptic fashion. The semi-continuouscentrifuges are capable of very high particle concentration factors e.g.100 to 1 and can also discharge MVL concentrate up to 80% PPV %. TheCentritech Lab III is semi-continuous as it has a constant feed with anintermittent concentrate discharge, every 10 seconds to 2 minutes. TheViaFuge Pilot has a constant feed which is interrupted every 2 to 10minutes by a rapid discharge cycle.

FIG. 10 depicts continuous buffer exchange and MVL concentration systemswhere the hollow fiber filters operate at lower particle concentrations,a condition where they have higher permeate rates. The centrifuge (seenin FIG. 10, component 10400), e.g. CARR ViaFuge Pilot, is used to do afinal concentration into the final product vessel (seen in FIG. 10,component 10500). In this system all three stages including the tanks(seen in FIG. 10, components 10100, 10200 and 10300) run at bothconstant volume and MVL concentration while the final tank (seen in FIG.10, component 10500) collects the concentrated buffer exchanged MVLsuspension.

FIG. 11 depicts a system for continuous buffer exchange and MVLconcentration including only centrifuges. Using typical process ratesfor the ViaFuge Pilot and the same feed conditions as the hollow fibersystem, provides a system that exchanges 99.9% of the buffer whileconcentrating the MVL suspension by a factor of 3 when using theparameters from Table 13. The saline inputs should only flow when theinput MVL stream is flowing.

Any tanks connected to these centrifuges must be large enough toaccommodate the intermittent nature of the centrifuge input and output.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A process for making an emulsion using acontinuous-flow emulsification system, wherein said emulsificationsystem comprises: a high shear mixer, comprised of a rotor and a stator;a recirculation loop, comprised of one or more recirculation lines andan exit line; a heat exchanger; one or more outlet lines, comprised ofat least one nozzle feed line; one or more continuous phase inlet lines;and a discontinuous phase inlet line; wherein the heat exchanger and thehigh shear mixer are connected together in the recirculation loop by theone or more recirculation lines, wherein an end of one of the one ormore recirculation lines connects to an inlet of the high shear mixerand an end of the exit line connects to an outlet of the high shearmixer; further wherein the one or more outlet lines and one or morecontinuous phase inlet lines are connected to the recirculation loop,wherein at least one of the one or more continuous phase inlet lines isdirectly connected to the one of the one or more recirculation lines andthe at least one nozzle feed line is directly connected to the exitline; further wherein the end of the discontinuous phase inlet line islocated less than ⅓^(rd) of a rotor diameter from the rotor and lessthan ⅓^(rd) of a rotor diameter from the rotation axis of the rotor andis in fluid communication with the rotor, said process comprising:feeding an organic discontinuous phase into the emulsification systemthrough the discontinuous phase inlet line and feeding an aqueouscontinuous phase into the emulsification system through one or morecontinuous phase inlet lines.
 2. The process of claim 1, wherein theorganic discontinuous phase comprises a triglyceride.
 3. The process ofclaim 1, wherein the emulsion comprises a triglyceride, surfactant, andwater.
 4. The process of claim 1 further comprising: passing the organicdiscontinuous phase through a hydrophobic sterilizing filter beforefeeding the aqueous discontinuous phase into the emulsification systemthrough the discontinuous phase inlet line; and passing the aqueouscontinuous phase through a hydrophilic sterilizing filter before feedingthe organic continuous phase into the emulsification system through theone or more continuous phase inlet lines.
 5. A process for making anemulsion using a continuous-flow emulsification system, wherein saidemulsification system comprises: a high shear mixer, comprised of arotor and a stator; a recirculation loop, comprised of one or morerecirculation lines and an exit line; a heat exchanger; one or moreoutlet lines, comprised of at least one nozzle feed line; one or morecontinuous phase inlet lines; and a discontinuous phase inlet line;wherein the heat exchanger and the high shear mixer are connectedtogether in the recirculation loop by the one or more recirculationlines, wherein an end of one of the one or more recirculation linesconnects to an inlet of the high shear mixer and an end of the exit lineconnects to an outlet of the high shear mixer; further wherein the oneor more outlet lines and one or more continuous phase inlet lines areconnected to the recirculation loop, wherein at least one of the one ormore continuous phase inlet lines is directly connected to the one ofthe one or more recirculation lines and the at least one nozzle feedline is directly connected to the exit line; further wherein the end ofthe discontinuous phase inlet line is located less than ⅓^(rd) of arotor diameter from the rotor and less than ⅓^(rd) of a rotor diameterfrom the rotation axis of the rotor and is in fluid communication withthe rotor, said process comprising: feeding an aqueous discontinuousphase into the emulsification system through the discontinuous phaseinlet line and feeding an organic continuous phase into theemulsification system through the one or more continuous phase inletlines.
 6. The process of claim 5, wherein the organic continuous phasecomprises an organic solvent and a neutral lipid.
 7. The process ofclaim 6, wherein the organic solvent is methylene chloride orchloroform.
 8. The process of claim 5 wherein the aqueous discontinuousphase comprises an acid and a therapeutic agent.
 9. The process of claim8, wherein the acid is phosphoric acid.
 10. The process of claim 8,wherein the therapeutic agent is bupivacaine.
 11. The process of claim5, wherein a portion of the emulsion is fed through the at least onenozzle feed line to a first fluid conduit of at least one atomizingnozzle apparatus, wherein said at least one atomizing nozzle apparatuscomprises: the first fluid conduit and a second fluid conduit eachhaving at least one entrance orifice and at least one exit orifice; afluid contacting chamber having a top comprising at least one entranceorifice connecting to the at least one exit orifice of the first fluidconduit and having a bottom comprising at least one exit orifice,wherein the at least one exit orifice of the second fluid conduitconnects to the top of the fluid contacting chamber; and a third fluidconduit, wherein the third fluid conduit annularly surrounds a portionof the fluid contacting chamber.
 12. The process of claim 11, whereinthe process further comprises applying a second aqueous phase to thesecond fluid conduit; and applying a gas to the third fluid conduit. 13.The process of claim 11, wherein between 50% and 99.99% of the emulsionexiting the high shear mixer is passed through the heat exchanger. 14.The process of claim 11, wherein between 80% and 99.99% of the emulsionexiting the high shear mixer is passed through the heat exchanger. 15.The process of claim 11, wherein between 1 mL/minute and 8,000 mL/minuteof the emulsion exiting the high shear mixer is fed through the at leastone nozzle feed line to the first fluid conduit of the at least oneatomizing nozzle apparatus.
 16. The process of claim 11, wherein between40 mL/minute and 100 mL/minute of the emulsion exiting the high shearmixer is fed through the at least one nozzle feed line to the firstfluid conduit of the at least one atomizing nozzle apparatus.
 17. Theprocess of claim 11 further comprising: providing the portion of theemulsion traveling to the at least one atomizing nozzle apparatusthrough the at least one nozzle feed line at a flow rate that is equalto a sum of the flow rate of the organic continuous phase through theone or more organic phase inlet lines and the flow rate of the aqueousdiscontinuous phase through the discontinuous phase inlet line.
 18. Theprocess of claim 5, further comprising forming emulsion droplets whichare on average less than 10 microns in diameter.
 19. The process ofclaim 5, wherein a length of one of the one or more recirculation linesannularly surrounds a length of the discontinuous phase inlet line. 20.The process of claim 5, wherein the organic continuous phase comprises atriglyceride.
 21. The process of claim 5, wherein the emulsion comprisesa triglyceride, surfactant, and water.
 22. The process of claim 5further comprising: passing the aqueous discontinuous phase through ahydrophilic sterilizing filter before feeding the aqueous discontinuousphase into the emulsification system through the discontinuous phaseinlet line; and passing the organic continuous phase through ahydrophobic sterilizing filter before feeding the organic continuousphase into the emulsification system through the one or more continuousphase inlet lines.